Myc Family Proteins: Molecular Drivers of Tumorigenesis and Resistance in Neuroendocrine Tumors

Abstract

Neuroendocrine cancers are a diverse and poorly understood collection of malignancies derived from neuroendocrine cells throughout the body. These cancers uniquely exhibit properties of both the nervous and endocrine systems. Only a limited number of genetic driver mutations have been identified in neuroendocrine cancers, however the mechanisms of how these genetic aberrations alter tumor biology remain elusive. Recent studies have implicated the MYC family of transcription factors as important oncogenic factors in neuroendocrine tumors. We take a systematic approach to understand the roles of the MYC family (c-MYC, n-MYC, l-MYC) in the tumorigenesis of neuroendocrine cancers of the lung, GI tract, pancreas, kidney, prostate, pediatric neuroblastoma, and adrenal glands. Reflecting the complexity of neuroendocrine cancers, we highlight the roles of the MYC family in deregulating the cell cycle and transcriptional networks, invoking cellular plasticity, affecting proliferation capacity, aiding in chromatin remodeling, angiogenesis, metabolic changes, and resistance mechanisms. Depicting the diversity of neuroendocrine cancers, we suggest new approaches in understanding the underlying tumorigenic processes of neuroendocrine cancers from the perspective of MYC.

1. Introduction

1.1. Neuroendocrine Cancers: Prevalence & Characteristics

Neuroendocrine Neoplasms (NENs) are a heterogenous collection of tumors that develop in neuroendocrine tissues. These tissues are key hormonal and signaling hubs that modulate organismal homeostasis¹. Although NENs constitute a small proportion of all solid tumors (as most tumors develop in epithelial tissues), their unique biology, cellular plasticity, and oncogenic landscape provide important insights into the mechanisms fueling tumorigenesis and metastasis. NENs often retain cellular features of neuroendocrine (NE) cells, enabling their identification in heterogeneous tumors that can contain a plethora of other cell types including epithelial, NE, fibroblasts, and immune cells. NENs are defined by their expression of common NE markers, including Synaptophysin (Syn or SYP) and Chromogranin A (CgA)^1,2. Characteristically, NENs present with neuronal features (i.e. dense core granules) while phenotypically mimicking endocrine cells by synthesizing and secreting monoamines as well as peptide hormones^1,2. Several NENs are diagnosed following carcinoid syndrome, a hypersecretion of amines and peptides, abnormally elevating hormone levels in patients¹. NENs tumors commonly contain poorly differentiated cells that promote cellular plasticity, fate changes, and expansion. These features contribute to widespread histological, molecular, and clinical heterogeneity amongst NEN lesions¹.

NE cells are distributed throughout the body and frequently make up small clusters of cells within larger epithelial organs. This enables NENs and metastatic lesions to be present in an array of sites (Figure 1). Primary tumors that develop from NE tissue are commonly found in the lungs (~25% of cases), gastrointestinal (GI) tract (~65%), and more rarely in the urogenital system, breast, skin, thyroid, respiratory tract, and central nervous system^1,3. Diagnosis at advanced stages is common. Around a fifth of patients with NENs present with local or distant metastases at the time of diagnosis^1,3,4. The majority of NEN tumors occur sporadically, however a small subset is caused by heritable genetic disorders, e.g., Multiple Endocrine Neoplasia 1 (MEN1) syndrome¹.

Figure 1: Anatomic Distribution of Human Neuroendocrine Neoplasms (NENs).

Occurrence sites of neuroendocrine neoplasms (NENs). Primary tumors that develop from neuroendocrine tissue are commonly found in the lungs (~25% of cases), gastrointestinal (GI) tract (~65%), and more rarely in the urogenital system, breast, skin, thyroid, respiratory tract, and central nervous system. Solid lines displayed indicate anatomical primary sites of tumor development, whereas dashed lines specify common sites of metastasis (lymph node, liver, spleen).

NENs can be further sub-divided into two subtypes: Neuroendocrine Tumors (NETs) or Large/Small-cell Neuroendocrine Carcinomas (NECs). Each subtype carries distinct characteristics that have important molecular and treatment implications. NETs are commonly well-differentiated, with low proliferative potential. These tumors are considered well organized with organoid-like properties and regulated growth patterns¹. Cells within these tumors have small nuclei and coarse chromatin compaction. In contrast, NECs are poorly differentiated, highly proliferative, and show disorganized growth patterns. These characteristics are accompanied by irregular cellular features and less secretory granules. Heterogenic by nature, NECs commonly contain cells that do not have NE features or markers, further complicating the tumor landscape¹.

1.2. Genetic Drivers of Neuroendocrine Cancers

1.2.1. Loss of Tumor Suppressors

Several genomic alterations have been implicated in the tumorigenesis of NENs. Commonly, tumor suppressor genes are inactivated or deleted, resulting in unrestricted proliferation, poor differentiation, and tumorigenesis. Small cell lung cancer (SCLC), a pulmonary NEN, has the near ubiquitous, concurrent loss of key tumor suppressor genes TP53 (p53 protein) and RB1 (pRB protein)^5–7. Lost in most cancer cells, transcription factor p53 senses genomic damage or aberrant proliferation signals. Based on the level of accrued damage, p53 promotes apoptotic pathways or cell cycle arrest for DNA damage repair⁸. p53 loss hinders the repair or elimination of damaged cells, supporting tumorigenesis⁹.

Tumor suppressor pocket protein pRB (retinoblastoma), along with related pRB family members p107¹⁰ and p130, serve as regulators of E2F transcription factors^11,12. E2Fs are a multifaceted family of transcription factors that interact with DNA at its consensus binding motif (TTTSSCGC; S can be C/G)^13,14. E2Fs heterodimerize with DP1/2 to enhance motif binding affinity in promoter regions and pRB family members^11,15,16. E2Fs diversely serve as both transcriptional repressors (E2F4, E2F5) and activators (E2F1, E2F2, E2F3)^11,14. Activating E2Fs heavily control the G1 to S cell cycle transition. E2F1, once accumulated in G1, binds to the promoters of S phase target genes (cyclin A/E) and proto-oncogenes (c-MYC) to drive cell cycle progression¹⁷.

CDK/cyclin complexes phosphorylate pRB at multiple serine and threonine residues, leaving pRB in an active hypo-phosphorylated state¹¹. In a hypo-phosphorylated state, pRB family members each preferentially bind certain E2F proteins throughout the cell cycle. pRB shows high affinity for activating E2Fs 1–3, specifically limiting progression from G1 into S phase and maintaining the integrity of the G1/S cell cycle checkpoint through its interactions¹⁸. Disrupting E2F-mediated transcriptional activation, the pRB family member binding domain sits in the transactivation domain of E2F proteins^18,19. Cyclin dependent kinases CDK4/6-cyclin D and CDK2-cyclin E reverse this effect by hyper-phosphorylating pRB^20,21, causing its dissociation from activating E2Fs. E2Fs 1–3 then induce the transcription of S phase target genes, beginning the progression of cells into S phase.

Both TP53²² and RB1 loss^23,24 are common among NE tumors. pRB loss effectively leaves cells with a defective G1/S checkpoint and loss of E2F negative regulation^23,24. SCLC murine models, patient tumors, and cell lines show DNA level gains in activating E2F family members^25–27, particularly E2F1²⁸ and E2F3²⁹, supporting uncontrolled proliferation.

The World Health Organization (WHO) reflects this common loss of TP53 and RB1 in NE tumors in their classification of digestive system NE tumors. The WHO recommends using TP53 and RB1 mutations to separate NECs from high-grade NETs. In a large molecular study, 64% of NECs compared to only 14% of NETS had TP53 mutations. However, loss or deletions of RB1 were common in both NECs and NETs (~30%)³⁰.

Tumor suppressor loss is common beyond TP53 and RB1. Pancreatic neuroendocrine tumors (PNETs) have been associated with inherited genetic syndromes caused by disruptions in the activity of tumor suppressor genes. Mutations within multiple endocrine neoplasia type 1 (MEN1), tuberous sclerosis (TSC1/2), neurofibromatosis type 1 (NF-1), and Von-Hippel Lindau (VHL) syndrome^31,32 have all been found in PNETs. Germline inactivating mutations in the MEN1 gene cause most familial PNETs where MEN1 encodes for MENIN, a transcription factor involved in chromatin remodeling, proliferation, and genome stability^31–33. Heightened PNET risk has been associated with loss of chromosome 11q, including loss of MEN1 as well as DNA repair genes (BRCA2, ATM)³¹. Among PNETs, MEN1 loss of function mutations resulted in hypermethylation of tumor suppressor genes HIC-1, RASSF1A, MGMT, MLH1, and CDKN2A, fueling tumorigenesis³¹. MENIN loss has also been associated with VHL mutations^31,34 that causes constitutive HIF activation and, consequently, uncontrolled angiogenesis. Sporadic PNETs frequently contain mutations in tumor suppressor genes DAXX and ATRX, tumor suppressors and chromatin remodelers in the histone H3.3 chaperone complex³³. DAXX or ATRX loss leads to chromosomal instability, reduced Histone 3 lysine 9 methylation (H3K9me), and aggressive PNET disease.

1.2.2. Activation of Oncogenes

In addition to tumor suppressor gene loss, activating mutations in oncogenes contribute to the development of NENs. The proto-oncogene RET, which encodes for a receptor tyrosine kinase, plays an active role in the development of the renal³⁵ and neuronal systems³⁶. Several human tumors of neural crest origin, including pheochromocytoma, pediatric cancer neuroblastoma (NB), and medullary thyroid cancer (MTC), have mutations within RET that result in its constitutive activation. Germline RET mutations cause NE tumor syndromes, MEN 2A and MEN 2B, and familial MTC³⁷.

Aberrant activation of Cyclin-Dependent Kinase 5 (CDK5), a non-canonical CDK family member, has been implicated in PNET growth. When complexed with p35 or p39 in NE cells, CDK5 functions in pancreatic islets to promote β-cell survival and hormone secretion. Aberrant activation of CDK5 by p25, a p35 cleavage product, results in PNET development in mice³⁸. Including but not limited to PNETs, several other potent oncogenes have emerged as potential drivers of growth and cell death evasion in human gastroenteropancreatic (GEP-NETs). Immunohistochemistry of 116 primary human GEP-NETs supported high expression of potent oncogenes BCL2, ERBB2/3, c-JUN, and c-MYC among these NE tumors³⁹.

The proto-oncogenic MYC family of transcription factors have emerged as diverse promoters of NEN tumorigenesis. Alteration in MYC levels reduces recurrence-free and overall survival of patients with high-grade pulmonary NE carcinomas⁴⁰.

In the pulmonary NET SCLC, 15–20% of patient tumors^6,25 exhibit mutually exclusive MYC family amplifications. MYC amplifications are associated with chemo-resistance, tumor progression, and shortened patient survival^41–44. In this setting, the MYC protein family members have complex roles in conjunction with four other transcriptional regulators (ASCL1, NeuroD1, YAP1 and POU2F3), contributing to the emergence of four SCLC subtypes: SCLC-A, SCLC-N, SCLC-Y, and SCLC-P)^45,46.

c-MYC has been implicated as a SCLC oncogenic driver⁴⁷ and biomarker^6,48 for targeted therapy. c-MYC promotes S phase cell cycle entry and proliferation through its activation of CDK4 and several cyclins^49,50. When overexpressed, c-MYC enables uncontrolled proliferation. In c-MYC driven SCLC Genetically Engineered Murine Models (GEMMs) (Rb1^Flox/Flox; Tp53^Flox/Flox; MycLSL^/+), the conditional overexpression of c-MYC yields highly proliferative and metastatic tumors, relative to Rb1^Flox/Flox Tp53^Flox/Flox SCLC GEMMs^23,42.

Through its diverse interactions with genes involved in growth and cell cycle control, MYC family members have been implicated in the temporal evolution, differentiation, oncogenesis, and chemo-resistance mechanisms of NE tumors (Figure 2). We break down the involvement of this family on NENs in this review.

Figure 2: Multifunctional Roles of MYC Proteins in Neuroendocrine Neoplasms (NENs).

The MYC transcription factor family (c-MYC, n-MYC, l-MYC) heterodimerizes with co-factor MAX at consensus E-boxes binding sites to promote the transcription of MYC target genes. In NENs, MYC has been linked to changes in transcriptional regulation, chromatin remodeling, the cell cycle, proliferation, metabolism, translation, ribosome biogenesis, angiogenesis, immune evasion, cellular plasticity, and resistance mechanisms.

1.3. The MYC Family of Transcription Factors

Under normal environmental conditions, cellular homeostasis is maintained by tightly controlling growth promoting signals and limiting cell cycle entry as well as progression. Cancer cells disrupt this regulation to promote cellular transformation and reprogramming, enabling deregulated growth⁵¹. As described above, NE tumors disrupt a number of tumor suppressor pathways to fuel transformation. In addition, these cancers also induce the expression or amplification of oncogenes to sustain chronic proliferation. These strategies are diverse, however the over-expression or amplification of the myelocytomatosis oncogene (MYC) transcription factor family is a common feature of many aggressive NE tumor subtypes (Figure 3).

Figure 3: MYC Transcription Factor Family Status Among Neuroendocrine Neoplasms (NENs).

Several neuroendocrine neoplasms (NENs) of different anatomical origins report an involvement of the MYC family of transcription factors (c-MYC, n-MYC, l-MYC) as oncogenic drivers. Basic, pre-clinical, and clinical work has been conducted in largely lung, pancreatic, and prostate NENs to understand the role of each corresponding MYC family member in neuroendocrine tumor development and resistance mechanisms. Many studies are actively investigating the impact of n-MYC on the pediatric malignancy neuroblastoma while NENs of the adrenal glands report a unique reliance on the MYC family binding partner MAX.

The pleiotropic MYC family of transcription factors, c-MYC, n-MYC, and l-MYC exhibit tissue specific expression patterns that provide important molecular clues into the role of each family member in tumorigenesis. c-MYC was first identified in 1982 as the human homolog of the transforming sequence in avian retrovirus MC29^52,53. c-MYC is the most widely expressed MYC family member and has strong oncogenic capacity in a diverse range of tumor types. c-MYC profiling in an array of tumors resulted in the identification of other MYC proteins. n-MYC, which is highly expressed in neural lineages, was identified as a c-MYC homolog using southern blots in neuroblastoma (NB)⁵². Similar probing of small-cell lung carcinoma (SCLC) identified l-MYC, based on its sequence conservation with c-MYC⁵². The MYC family is highly conserved. Both n- and l-MYC show ~80% amino acid sequence homology with c-MYC and retain many of the key features of c-MYC. This includes the 3-exon structure of c-MYC, basic helix-loop-helix and leucine zipper region (BR-HLH-Zip), as well as both Myc box domains (MbI/II)^52,54.

1.3.1. Multifunctional Roles of MYC

The MYC family of proteins are transcription factors that directly bind to defined E-box sequences within the promoter regions of their targets⁵⁵. MYC binding primes genes for active transcription. This interaction is key for altering the chromatin and transcriptional environment for genes⁵⁵. MYC proteins commonly bind to promoters close to the Transcriptional Start Site (TSS) to maximize its capacity to amplify transcription. Analysis of genes bound by MYC in human cells shows that MYC is an important regulator of cell cycle genes^56,57.

MYC has been shown to interact with a number of co-factors that support MYC activity. MYC Associated Factor X (MAX), another bHLH-ZIP protein, and likely distant MYC homolog⁵⁸, heterodimerizes with MYC, promoting MYC binding fidelity to high priority E-box sites containing an extended consensus sequence, ACCACGTGGT⁵⁹.

MYC’s Amplification Patterns, Promiscuous Behavior, & Role in Transcriptional Deregulation

The MYC family is suggested to act as more of a global enhancer of transcription, accelerating transcriptional activation through releasing paused RNA polymerase at relatively any open promoter regions. However, MYC occupancy also seems to be determined by relative MYC levels. Somewhat following a strict amplification model, MYC at moderate protein levels does bind to its high-affinity E-box containing binding sites. Yet not all occupied MYC binding sites demonstrate transcriptional activation, likely being modulated by other transcription factors either locally or distally from affected enhancers. MYC overexpression further enables this factor’s binding preference to include lower-affinity sites, invading enhancers and promoters. This promiscuity fuels widespread transcriptional changes and cellular stress, ultimately triggering cell cycle arrest or apoptosis. However, tumor suppressor loss, including p53 and pRB, enable cells to bypass these protective processes, ultimately contributing to tumorigenesis^60,61.

MYC’s Roles in Chromatin Remodeling

The MYC protein family commonly complexes with Bromodomain proteins (BRD), including BRD4^62,63. BRD proteins are chromatin remodelers⁶⁴, and via this interaction with MYC, are able to facilitate chromatin changes around MYC-driven genes that are conducive to transcription. An ever-growing number of chromatin modifying enzymes have been linked to MYC activity, suggesting that MYC not only induces transcription, but sets up transcription factories to maximize gene expression^65,66.

MYC’s Roles in Cell Cycle Progression & Proliferation

MYC has a key role in amplifying the capacity of existing transcription factors at gene promoters. The best characterized example is MYC and activator E2Fs (E2F1-E2F3). E2F1-E2F3 are central cell cycle regulators⁶⁷ and bind tightly to promoters containing a conserved E2F binding domain close to the TSS. In this role, E2F activity is critical for the gene expression required for the progression through the G1/S checkpoint of the cell cycle^67,68. In tumors with MYC amplification and/or MYC overexpression, abundant MYC (and its associated co-factors) directly bind to E2F at promoters⁶⁹. This facilitates rapid MYC loading, inducing strong transcriptional upregulation⁶⁹ and fueling cell cycle progression, proliferation, and tumorigenesis.

MYC’s Roles in Metabolism

As an amplifier of gene expression, the MYC family of proteins promotes the homeostatic expression of “house-keeping genes” with open chromatin structures. MYC’s canonical E-box binding sites are enriched within these genes, including those required for ribosome assembly and mitochondrial biogenesis⁶¹. Supporting ribosome biogenesis and growth, MYC promotes the expression of the majority of glycolytic genes^70,71, including lactate dehydrogenase A (LDHA)⁷², hexokinase 2 (HK2), and glucose transporters⁷³. These glycolytic genes report phylogenetically conserved MYC E-boxes, suggesting direct and retained MYC regulation⁷⁴.

Aiding cancer cell survival under hypoxic stress, these MYC binding sites also overlap with those of hypoxia inducible factor 1 (HIF-1), allowing for the stimulation of glycolytic genes under hypoxia and increasing anaerobic glycolysis^75–78. When upregulated, the MYC family drives tumorigenesis by transcriptionally reprogramming metabolism, facilitating the necessary production of macromolecules required to sustain uncontrolled proliferation and growth. Through this mechanism, c-MYC can promote aerobic glycolysis, contributing to the Warburg effect in tumors, and enhance glucose uptake, lactate production, and the glycolytic intermediates required for nucleotide, amino acid, and lipid biosynthesis processes⁷⁹.

Along with its regulatory roles in glycolysis, MYC also has been shown to promote glutoaminolysis in proliferating cells. MYC both sensitizes cells to death by glucose-deprivation and confers a dependency or addiction to glutamine through upregulating genes, including glutaminase (GLS), to drive TCA cycle activity^80–82. GLS has been implicated in cancer cell metabolism due to its role in converting glutamine to glutamate⁸³. GLS inhibitors, namely BPTES, increase survival in vivo in both MYC-inducible liver cancer and renal cell carcinoma^83–85.

MYC regulates genes involved in mitochondrial biogenesis and activity, e.g., PGC1β and TFAM^79,86,87. Along with this, MYC has been implicated in nucleotide metabolism^88,89, specifically by inducing the expression of CAD⁹⁰ and regulating DHODH⁸⁸, both of which are involved in pyrimidine synthesis. c-MYC has further been demonstrated to promote lipid metabolism during cell cycle entry and lymphomagenesis^79,91,92.

Overall, altered MYC levels promote metabolic reprogramming and macromolecule production integral for elevated tumor growth rates. Therapeutically, cancer cells with increased MYC activity are sensitive to nutrient withdrawal and the inhibition of MYC-driven tumor-promoting metabolic pathways^79,93.

MYC’s Roles in Translation & Ribosome Biogenesis

Ribosome biogenesis is a tightly controlled process. The generation of ribosomal RNA (rRNA) that forms the basis of ribosome assembly requires the accurate transcription, processing, and modification of rRNA. This process is mediated by a plethora of factors including RNA polymerases, transcription factors, small nucleolar RNAs, and non-ribosomal proteins. Ultimately, mature rRNA and ribosomal proteins assemble to form the 40S and 60S ribosomal subunits^94,95.

Mature ribosome levels are controlled by signal transduction pathways that inform cells on their growth conditions and modulate ribosomal components’ activities and expression. Unsurprisingly, this careful balance between these factors and their growth signaling pathways are often disrupted in tumors. Activating mutations within growth signaling pathways and Pol I transcription machinery are common in cancer, suggesting a prevalent upregulation of rRNA synthesis in tumorigenesis^95,96.

The MYC family, along with the potent PI3K-AKT-mTOR signaling pathway, promote ribosome biogenesis in several ways^95,97. Overexpressed MYC promotes mature ribosome generation and increases the protein production potential of cancer cells^98,99. MYC directly binds and transcriptionally controls the expression of ribosomal subunits and rRNA, as well as the factors needed for rRNA processing, ribosome nuclear export, and mRNA translation initiation⁷⁰. Specifically, MYC activates Pol II and Pol III transcription of ribosomal proteins and 5S rRNA^99,100. In the nucleolus, MYC upregulates rDNA transcription by promoting the assembly of the Pol I preinitiation complex and enhancing rDNA transcription factor expression (UBF, SL1, TIF-1A, POLR1B)^101–103. mTOR further regulates TIF-1A that associates with Pol I, affecting rRNA synthesis¹⁰⁴.

Concurrent MYC and PI3K-AKT-mTOR hyperactivation are associated with treatment resistance and tumor aggressiveness in several tumor types^95,105,106. More studies are required to elicit the mechanisms underpinning the synergism between MYC and PI3K-AKT-mTOR in ribosome biogenesis, tumorigenesis, and treatment resistance⁹⁵. Interestingly, MYC-driven tumorigenesis remains dependent on its capacity to modulate and effectively increase protein synthesis. Haploinsufficiency in ribosomal proteins decreases MYC-induced tumor growth, suggesting mature ribosome levels are key to MYC’s oncogenic capabilities⁹⁸.

MYC’s Roles in Angiogenesis

Angiogenesis, or the outgrowth of new blood vessels, is essential for tumor growth. MYC activity has been shown to stimulate angiogenesis to support tumorigenesis¹⁰⁷. Despite the lack of a mechanism, previous studies in neuroblastoma, lymphoma, a fibroblast xenograft, and the skin indicated an association between MYC activation and angiogenic capacity^108–111.

During normal development, c-MYC functions in vascular and hematopoietic development by promoting angiogenic factors. The embryonic lethality of c-MYC^−/− mice has been linked to defects in vasculogenesis and erythropoiesis. In this setting, c-MYC activity is required for the induction of the pro-angiogenic factor, Vascular Endothelial Growth Factor (VEGF)¹¹². Using involucrin c-MYC-ER^TAM transgenic mice, MYC was subsequently shown to promote angiogenic capacity during hypoxia, again through the VEGF regulation¹⁰⁷. MYC has also been shown to bind and silence inhibitors of angiogenesis including thrombospondin-1 (tsp-1), a negative regulator of angiogenesis¹¹³. It was later shown that MYC binds to a non-canonical E-box in the tsp-1 promoter region with low affinity and blocks de novo RNA synthesis, downregulated tsp-1 through increasing its mRNA turnover¹¹⁴.

MYC’s Roles in Immune Evasion

The MYC family has been implicated in tumor immune evasion, supporting tumor survival and immunotherapy resistance. MYC’s role in immune evasion has been linked to a number of mechanisms, ranging from suppressing MHCI expression to upregulating inhibitory cytokines or immune checkpoint proteins, PD-L1 and CD47¹¹⁵.

Elevated MYC levels have been associated with reduced CD8⁺ T cell infiltration, insensitivity to immune-checkpoint blockade, and increased expression of the immune-checkpoint proteins among 33 different human cancer types. In hepatocellular carcinoma, MYC represses MHC class I expression, hindering antigen presentation and the T-cell response. A combinatory therapeutic approach of PDL1 and CTLA4 reversed this observed MYC-driven immune suppression. This approach led to the recruitment of CD40 and MHCII expressing pro-inflammatory, antigen-presenting macrophages that when depleted diminished the combination approach’s anti-tumoral effects¹¹⁶. Similarly, in MYC-elevated triple-negative breast cancer (TNBC), the inhibition of oncogenic MYC promoted the expression of the immune-related hallmark gene set and tumor-infiltrating T-cells. A CRISPR-mediated screening approach identified that MYC can repress the expression of innate immune modulator, STING, to promote immune evasion. This repression was associated with PD-L1 blockade resistance in TNBC murine models. In immune-cold TNBC, MYC inhibitor MYCi975 combined with anti-PD-L1 immunotherapy produced an antitumoral effect¹¹⁷.

Additional support for these observations has been found in MYCN amplified neuroblastoma or c-MYC amplified melanoma. Treating cells from these tumors with bromodomain inhibitors (I-BET726 and JQ1) to indirectly affect MYC activity resulted in cell cycle arrest and enhanced immunogenicity. This work supports the development of tumor vaccines in combination with checkpoint inhibition in neuroblastoma and melanoma tumors¹¹⁸. These findings also suggest that targeting MYC-associated pathways may promote tumor immune responses in cancer patients and improve patient outcomes¹¹⁹.

MYC’s Roles in Cellular Plasticity & Resistance Mechanisms

Intratumoral heterogeneity has been shown to be driven in part by cellular plasticity. This plasticity or stemness has been associated with therapeutic resistance and recurrent disease^120–122. Multiple studies have associated N-MYC and c-MYC in the NE trans-differentiation observed in prostate adenocarcinoma, driving its progression to an aggressive NE phenotype^123–126. Showing similarities, another study reported that c-MYC has a regulatory role in the ductal-NE plasticity observed in pancreatic ductal carcinoma, contributing to chemoresistance and diminished survival. Drawing several parallels, c-MYC targeted genes in pancreatic ductal carcinoma were also enriched in neuroendocrine-differentiated prostate cancer. Moreover, c-MYC loss in pancreatic ductal carcinoma reduced the expression of NE markers¹²⁷. Within the context of MYC-driven NE tumors, cellular plasticity and NE differentiation are likely contributing to observed chemoresistance, aggressiveness, and tumor survival.

2. Neuroendocrine Tumors by Body Location/System

2.1. Pulmonary NENs

The lung is the second most common primary site for NENs, constituting ~20% of all lung cancers¹²⁸. As with gastrointestinal NENs, lung NENs are subdivided by grade which encompasses histology, NE gene expression, proliferation index, and frequency of apoptosis. SCLC and large-cell neuroendocrine cancer (LCNEC) are classified as high-grade NENs. Pulmonary carcinoid tumors (PCs) are considered low grade NENs that can be further divided into atypical (AC) and typical (TC) subtypes. Amplifications of MYC family genes MYC, MYCN, and MYCL are more common in the high-grade lung NETs¹²⁹, and as in other cancers, these amplifications portend a worse overall prognosis^129–132 (Figure 4).

Figure 4: MYC Family Amplifications in Pulmonary Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) commonly arise in the lung. These tumors are subdivided by histologic grade. In ascending order these include typical carcinoids (TC), atypical carcinoids (AC), large cell neuroendocrine cancer (LCNEC), and small cell lung cancer (SCLC). Lung NETs constitute 20% of all lung cancers, with SCLC by far the most common. Lung NET grade correlates directly with frequency of MYC family gene amplification and level of expression, and inversely with overall survival.

SCLC and LCNEC share similar genomic and transcriptional features but have distinct stage-specific management recommendations. LCNEC is classified along with lung adenocarcinoma and squamous cell lung cancer as a non-small cell lung cancer (NSCLC), meaning that LCNEC patients with limited metastases (early stage) can still be cured with surgery. In contrast, SCLC has commonly metastasized at diagnosis limiting the utility of surgery to rare cases with small solitary lesions and always followed by adjuvant chemotherapy. SCLC is the highest-grade lung NEN. These cells have distinct features including predominant small cell diameter (<3 lymphocytes), scant cytoplasm, indistinct nucleoli, frequent mitoses and spontaneous apoptosis¹²⁸. In contrast, LCNEC cells have an abundant cytoplasm and conspicuous nucleoli. Despite these cellular differences, both SCLC and LCNEC express traditional NE markers, such as Syn/SYP, CgA, NCAM, and INSM1¹²⁸. SCLC and LCNEC are the most common lung NENs (15% and 3% of lung cancers, respectively)¹³³. Both are strongly associated with smoking, commonly present with metastases, and carry poor overall prognoses, with 5-year survival rates of 17% for LCNEC and 5% for SCLC^133–135.

In contrast to SCLC and LCNEC, PCs are low-grade malignancies with infrequent mitoses (<10 mitoses per 2 mm²)¹²⁸. In addition to traditional NE markers, PCs preserve expression of genes that characterize differentiated pulmonary neuroendocrine cells (PNECs), such as somatostatin receptors (SSTRs). PCs are divided into AC and TC based on proliferation rate^136,137. Metastasis is more frequent in AC (20% of cases) than TC (10%)^136,138. As with many solid tumors, PC differentiation is anticorrelated with grade, with TC presenting with higher SSTR expression and more frequently carcinoid syndrome caused by inappropriate hormone secretion. PCs constitute less than 2% of all lung cancers, however TC is (8–10 times) more common than AC and arises more frequently in non-smokers (20–40%). Their prognosis is much better than aggressive lung NENs, with 5-year survival rates of 64% for AC and 84% for TC¹³³. Although PCs are NE, their biology is distinct from SCLC and LCNEC, and even in metastatic and fatal cases, transformation of a PC into a more aggressive NEN is almost never observed.

2.1.1. MYC in Small Cell Lung Cancer

Amplifications of MYC and its paralogs, MYCL and MYCN, were the first recurrent genomic alterations to be identified in SCLC^139–142, preceding the discoveries of biallelic loss of RB1 and TP53 that have become the genomic hallmarks of the disease^143,144. These MYC genes are highly expressed in SCLC, with or without amplification, and their expression in both tumors and benign tissues are mutually exclusive^145–147. MYC family amplifications are present in 15–20% of SCLC tumors at diagnosis^7,25,148,149, and additional amplifications can be acquired after chemotherapy^43,150–153. These amplifications are frequently numerous, exceeding 100 copies per cell, and usually arise on extrachromosomal DNA (ecDNA)^132,139,154. Mutations in MYC-regulatory factors such as MAX, MGA, and BRG1 are also recurrent¹⁵⁵, underscoring the importance of MYC family activity in SCLC.

GEMMs demonstrate roles for MYC family genes in SCLC tumorigenesis. Murine small cell lung carcinoma (MSCLC) can be generated by inactivation of Rb1 and Trp53 with tumor formation after 11–12 months¹⁵⁶. During this latency, spontaneous amplifications of Mycl arise frequently^157,158, and overexpression of c-Myc, n-Myc, or l-Myc can all accelerate tumorigenesis^42,159–161. In-frame fusions between RLF and MYCL are found in approximately 5% of patients, and when modeled in mice these fusions also accelerate SCLC tumorigenesis^148,162,163. RLF-MYCL is the first oncogenic gene fusion to be described in SCLC.

MYC family members also play a major role in resistance to the only drugs that are consistently effective in SCLC: DNA-damaging chemotherapies. For first-line therapy, all SCLC patients receive doublet chemotherapy with a platin (cisplatin or carboplatin) plus a topoisomerase inhibitor (etoposide or irinotecan)¹³⁴. For patients with disease restricted to one hemithorax, radiation is added to chemotherapy. For all other patients, immune checkpoint inhibitors are added and provide durable benefit for a small number of patients. This approach has remained largely unchanged for the past 40 years because it is highly effective, with response rates in excess of 70%. Unfortunately, relapse is nearly inevitable, and results in acquired resistance that extends beyond the platins and topoisomerase inhibitors to encompass most antineoplastic agents^164,165. Acquired cross-resistance to chemotherapy renders SCLC untreatable and rapidly fatal, and discovery of the molecular drivers remains a priority in SCLC research¹³⁴.

Early studies of SCLC cell lines revealed that MYC family gene amplifications are more frequent in relapsed SCLC^43,44. This led to the hypothesis that MYC family gene amplifications can drive chemoresistance. Recent experiments in patient-derived xenograft (PDX) models of SCLC support this idea. PDX models accurately recapitulate SCLC patient responses to diverse clinical regimens^166–169. In PDX models derived from untreated patients, ectopic expression of n-MYC or l-MYC can drive resistance to cisplatin and etoposide treatments¹⁶¹. In longitudinal PDX models derived before and after treatment, extreme ecDNA amplifications of MYC family genes (ecMYC/L/N) are acquired frequently after relapse and drive cross-resistance to multiple chemotherapy regimens¹⁵². The mechanisms by which ecMYC/L/N amplifications arise and confer cross-resistance are not known but are under active investigation.

As of 2025, the dated systemic treatment approaches for SCLC fail to account for the extensive observed diversity in SCLC histology and emerging plastic disease subtypes. Early studies of SCLC cell lines revealed a “variant” subtype distinguished from the “classic” majority by increased cytoplasm, occasional nucleoli, and decreased expression of NE markers^170,171. These subtypes partitioned MYC family gene expression, with MYCL predominant in classic SCLC, MYC in variant SCLC, and MYCN observed in both subtypes^141,145,171. More recently, these subtypes have been associated with neurodevelopmental transcription factors, with ASCL1 expressed in classic SCLC and NEUROD1 and POU2F3 expressed frequently in variant SCLC^45,172,173. The association between the “ANP” (ASCL1/NEUROD1/POU2F3) transcription factors and MYC family genes is not merely correlative. Ectopic MYC expression during SCLC tumorigenesis, or in established tumors, drives a transition from classic to variant morphology and from ASCL1 to NEUROD1 expression, and may affect MYCL expression since it is a direct transcriptional target of ASCL1^{172,174–176}. The relationships between ANP transcription factors, MYC paralogs, and NE expression programs are current areas of active research, with particular focus on the development of biomarkers for individualized therapy.

It remains correlative and unclear mechanistically how MYC is affecting SCLC resistance, subtype plasticity, and tumorigenesis. However, mechanistic studies are beginning to tease out the role of MYC in SCLC’s metabolic dependencies. MYC-driven SCLC relies on arginine-regulated pathways, e.g., polyamine biosynthesis, as well as the mTOR pathway. In GEMMs, human cell line xenografts, and PDXs of MYC-driven tumors, arginine depletion through pegylated arginine deiminase (ADI-PEG 20) repressed tumor growth and improved survival. This suggests a new therapeutic strategy in MYC-elevated SCLC¹⁷⁷.

Interestingly, SCLC exhibits a high sensitivity to Aurora A/B kinase inhibition (AURKAi/Bi)^{41,42,147,178–181}. Several reasons have been connected mechanistically to this sensitivity, including the genetic loss of RB1. As mentioned above, RB1 loss is common in SCLC. The loss of RB1 confers a strong dependency on AURKA/B for survival^178–180. Notably, high MYC expression, present in SCLC, is sufficient to sensitize RB1-mutants to the AURKA/Bi^{41,42,147,178,181}. SCLC with high MYC expression report suppressed tumor progression and increased survival when Aurora kinase inhibition is combined with chemotherapy⁴². MYC amplifications can lead to overexpressed MYC protein which in turn upregulate several genes involved in cell cycle progression, proliferation, and metabolism. MYC is known to induce AURKA transcription while AURKA has been known to regulate mitosis and SCLC tumorigenesis¹⁸². Aurora kinases A and B expression are upregulated by c-MYC and have been found to be essential for the tumor maintenance¹⁸³.

Although the mechanistic details of AURKA/B function remain incomplete in SCLC, data from lymphoma show that AURKA is a direct transcriptional target of MYC, which in turn supports tumorigenic phenotypes fueled by MYC. In this setting, AURKA/Bi trigger mitotic arrest, aneuploidy/polyploidy and apoptosis in MYC-amplified tumors¹⁸³.

2.1.2. MYC in Large Cell Neuroendocrine Carcinoma

Like SCLC, LCNEC is a highly heterogeneous tumor, and this heterogeneity extends to the expression and amplification of MYC family genes. Unlike SCLC, the dividing line appears to be the RB1 genotype. Although TP53 loss is nearly as frequent in LCNEC as in SCLC (71–92%), biallelic inactivation of RB1 and TP53 is found in only 27–40% of tumors^133,184,185. The recurrent mutations in RB1 wild-type LCNEC resemble other NSCLCs, with frequent inactivation of the tumor suppressors KEAP1, STK11, and CDKN2A, and activating mutations in RAS or RAF family genes (usually KRAS). Beyond co-mutation profiles, the LCNEC subtypes have distinct histology and gene expression patterns, with higher proliferation rates observed in the SCLC-like RB1 mutant tumors, but surprisingly, higher ASCL1 and NE expression levels in the NSCLC-like RB1 wild-type tumors. As with SCLC, LCNEC can harbor amplifications in all three MYC family genes, and the overall frequency is similar (13–22%). However, MYCL amplifications are found exclusively in the SCLC-like subtype, despite the lower level of ASCL1 expression, whereas MYC and MYCN amplifications are more common among the NSCLC-like tumors. These distinctions suggest that LCNEC represents a blend of at least two malignancies with distinct tumor suppressor and oncogene dependencies, and a major focus of LCNEC research is to translate these distinctions into genotype-directed therapies.

2.1.3. MYC in Pulmonary Carcinoids

MYC family amplifications are infrequent in PCs, and expression of these genes is markedly lower than in LCNEC and SCLC^186,187. When amplifications are observed, they are almost always MYCL amplifications arising in ACs¹³⁰. Beyond the MYC genes, the recurrent genomic alterations observed in PCs are generally distinct from those observed in LCNEC and SCLC. Common alterations in PCs occur in components of the SWI/SNF chromatin remodeling complex (40% of TC and 22% of AC), the MEN1 tumor suppressor (47% of TC and 70% of AC), and PIK3CA, which encodes the catalytic subunit of PI3K (13% of TC and 39% of AC)^186,188,189. In contrast, inactivating alterations of the tumor suppressors TP53 and RB1, which are frequent in LCNEC and nearly universal in SCLC, are rarely observed in PCs¹⁸⁶. Since tumor suppressor loss mitigates MYC-induced apoptosis and senescence, preserved function of p53 and pRB may underly the low frequency of MYC amplifications in PCs^190–192.

2.2. Gastroenteropancreatic NENs

The digestive system remains the most common site for the development of NENs, accounting for ~67% of NENs where most NENs are pancreatic¹⁹³. Interestingly, it has been estimated the MYC transcription factor family is amplified in as high as fifty percent of gasteroenteropancreatic (GEP) NECs¹⁹⁴. Furthermore, drugs against n-MYC’s interacting partners, including EZH2¹⁹⁵ and Aurora Kinases, report some success in preclinical studies and clinical development³⁰.

2.2.1. MYC in Gastrointestinal NECs (GIS-NECs)

Neuroendocrine carcinomas in the GI system (GIS-NECs) are relatively rare among cancer types but remain aggressive malignancies, distinct from other site-specific GI tract neuroendocrine tumors (GIS-NETs). GIS-NECs share a hypermethylation of the promoter region of the SOX2 gene, important for NE differentiation¹⁹³. Unlike the majority of GIS-NECs which have significantly mutated TP53, RB1, and MYC in WGS and WES studies, most non-pancreatic GIS-NECs (Nonpanc-NECs) with an intact RB1 exhibit a unique mutually exclusive amplification of MYC or Cyclin E1 (CCNE1), along with an inactivation of the Notch family¹⁹³. Despite these findings, further studies are needed to define molecularly how MYC is contributing to GIS-NECs of both pancreatic and non-pancreatic origins.

2.2.2. MYC in Pancreatic NENs (Islet-cell Carcinomas)

MYC in Pancreatic Neuroendocrine Tumors (PNETs)

Pancreatic neuroendocrine tumors (PNETs) are rare, slow growing malignancies that when localized have a relatively good prognosis with ~70% survivorship at 5 years. Tumors that have metastasized, largely to the liver (via the lymphatic system and lymphangiogenesis¹⁹⁶), significantly reduce the prognosis of patients and contribute to patient mortality rates. PNETs requiring a portal vein resection during pancreatectomies also report reduced survivorship¹⁹⁷. Several therapeutic approaches have been tested from chemotherapy, somatostatin analogs, and radiotherapy to predominantly used surgical resection^197,196.

Studies have begun to tease out the instrumental molecular factors contributing to lymphangiogenesis (the formation of new lymphatic vessels) in PNETs, realizing its key role in supporting tumor metastasis through the lymphatic system and contribution to poor patient survival. Nevertheless, it remained unclear how the observed transcription factor c-MYC was contributing to PNET lymphangiogenesis, cellular plasticity, and growth signaling.

Using PNET cell lines, it was revealed that Vascular Endothelial Growth Factor C (VEGFC) expression and secretion was notably higher in PNET cell lines with high c-MYC expression. Mechanistic work showed that c-MYC directly binds to a defined E-box in the VEGFC promoter and mediates its transcriptional upregulation, contributing to lymphatic endothelial cell (LEC) and PNET cell secretion¹⁹⁶. This sets up an intrinsic and extrinsic signaling cascade the promotes tumor growth and likely alters the tumor microenvironment (TME). Experimental testing of the role of c-MYC in PNET xenograft models showed that elevated c-MYC was sufficient to promote tumorigenesis and metastasis of PNET tumors¹⁹⁶. This effect was at least in part via VEGFC upregulation for treating these MYC mice with VEGFC-neutralizing antibodies in combination with mTOR inhibitors reduced VEGFC expression and LEC tube formation, slowed tumor development, and suppressed metastasis¹⁹⁶. In this context, c-MYC uniquely contributes to promoting metastasis by upregulating VEGFC signaling, fueling malignant features of PNETs and priming the TME for tumor outgrowth¹⁹⁶.

MYC in Pancreatic Ductal Adenocarcinomas (PDACs)

Unlike PNETs, Pancreatic Ductal Adenocarcinomas (PDACs) are relatively aggressive tumors¹⁹⁷, associated with very poor patient prognosis and cigarette smoking¹⁹⁸. PDACs arise from epithelial cells, frequently containing ductal-NE features. These cells are poorly differentiated and retain features of both lineages, providing an ideal setting for uncoordinated cell growth, expansion, and tumor development. KRAS-activation is an important driver of PDAC tumorigenesis, appearing in approximately 90% of PDAC cases¹⁹⁸, however new studies show that c-MYC also contributes to PDAC-NE lineage plasticity and intratumoral heterogeneity¹²⁷.

Using classical PDAC murine models (like the KC model: LSL-Kras^G12D; Pdx-1-Cre), c-MYC enrichment was observed at NE genes and c-MYC loss diminished the survival of PDAC cells, reducing the capacity of cells to retain ductal-NE features. This resulted in cells having strong ductal features and sensitized these tumors to gemcitabine treatment¹²⁷.

The data supports that PDACs exposed to chemotherapeutic pressure have higher NE markers¹²⁷. Paralleling the reported NE transformations in small cell neuroendocrine prostate cancer as well as lung/prostate adenocarcinomas^{126,199–201}, this has supported the concept that MYC may be involved in an adaptive drug resistance mechanism. MYC may drive PDAC lineage plasticity, resulting in intratumoral heterogeneity, helping PDAC overcome therapeutic pressures.

2.3. Genitourinary Tract NENs

2.3.1. MYC in Renal NENs

Considering the classifications of renal tumors described by WHO^202,203, there are 2 major subtypes of primary renal NENs: poorly differentiated neuroendocrine carcinomas (large- and small-cell subtypes) and well-differentiated NETs (carcinoid and atypical carcinoid tumors)²⁰³. Among these tumor types, there is limited but emerging evidence about the roles of MYC.

MYC in Poorly Differentiated Neuroendocrine Carcinomas (PDNECs) of Kidney

Poorly differentiated neuroendocrine carcinomas (PDNECs) of the kidney are very rare tumors with a mere 42 cases discussed in literature. It has been suggested that this malignancy originates from a multipotent stem cell capable of NE and exocrine differentiation. Although this remains unclear, this origin provides some explanation for the mixture of neuroendocrine and epithelial features observed in PDNECs^204–206.

Limited genomic profiling has been performed on these tumors. In agreement with other NE tumors, loss of TP53 and MYC gene amplifications were found to be present. Although molecular testing of how MYC functions in these tumors has not yet been conducted, MYC amplifications are present in tumors with chromosomal instability (CIN)²⁰⁴. This suggests that MYC may support high proliferation rates and genome evolution to sustain tumorigenesis²⁰⁴.

MYC in Primary Renal Well-Differentiated Neuroendocrine Tumors (Carcinoids)

The mutational profile of primary renal well-differentiated neuroendocrine tumors (carcinoids), or as discussed here as renal NETs, remain indistinct with most affected pathways being non-specific to renal NETs²⁰³. Neuroendocrine cells in normal renal tissue have yet to be identified, further complicating the cellular origins of renal NETs^207,208. Profiling studies are limited, however concurrent occurrences of renal NETs with the development disorder horseshoe kidney and teratomas have been identified. Considering 9 renal NETs, in a Next Generation Sequencing panel of genes associated with cancer, CDH1 and TET2 were identified as the most frequently mutated genes amongst the tumors. Mutations within MYC, AKT3, PIK3R2, ROS1, and BCR were also identified in 2 cases. These mutations tentatively suggest a role of MYC regulation and PI3K-AKT-mTOR signaling pathway in renal NETs, mirroring lung neuroendocrine carcinomas²⁰³. Nevertheless, more molecular studies are required to determine the role of and reliance on MYC in these rare tumors.

2.3.2. MYC in Prostate NENs

Prostate Adenocarcinoma (PAC) & Castration-Resistant Prostate Cancer (CRPC)

Prostate Adenocarcinoma (PAC) represents the most common form of prostate cancer (PCa) while PCa remains a global leading cause of male cancer-associated death²⁰⁹. The prostate is functionally reliant on androgen hormones whose actions are mediated by the androgen receptor (AR), a hormone activated transcription factor²¹⁰. Understanding this reliance, advanced or metastatic PCa is treated primarily with androgen deprivation therapy (ADT)²¹⁰ to suppress the production of testosterone and PCa growth. Resistance to primary AR targeted therapies enzalutamide and abiraterone^209,210 is common, leading to castration-resistant prostate cancer (CRPC) (Figure 5).

Figure 5: n-MYC’s Role in the Transition from Castration-Resistant Prostate Adenocarcinoma (CRPC) to Neuroendocrine Prostate Cancer (NEPC).

(A) Androgen responsive pancreatic cancer cells are known to develop androgen deprivation therapy resistance via several mechanisms (AR amplification, AR mutants, etc.). The resulting castration resistant pancreatic cells (CRPC) experience an increase in n-MYC and EZH2, promoting neuroendocrine prostate cancer (NEPC) development. Through its interactions with EZH2, n-MYC drives the NEPC phenotype and molecular program. As a result, n-MYC is able to abrogate AR signaling, enhance AKT activity, sensitize cells to EZH2 inhibitors, and support Aurora-A interactions. (B) The Rickman lab pre-clinical mice model mimics the molecular program of NEPC in vivo and reports similar histology, supporting the study into n-MYC and EZH2 emerging as oncogenic drivers in the NEPC phenotype. Adapted from:^124,214

MYC in Neuroendocrine Prostate Cancer (NEPC)

Approximately 10% of patients undergoing ADT with enzalutamide or abiraterone subsequently develop small cell neuroendocrine prostate cancer (NEPC)¹²⁴. In vitro and in vivo studies support trans-differentiation or conversion to a NE state as a potential mechanism of treatment resistance to androgen withdrawal^126,199,200. This process appears analogous to epithelial NSCLC tumors undergoing a conversion to more NE-like SCLC features²⁰¹.

NEPC is an aggressive, lethal subtype of PCa that arises following ADT for PAC and subsequently after CRPC¹²⁶ (Figure 5). An overlap of genomic alterations is shared between NEPC and CRPC, suggesting a transition from CRPC to NEPC. Similar to CRPC, NEPC reports ~50% of ERG rearrangements and ~63% of PTEN deletions¹²⁴. Unlike CRPC, NEPC expresses several NE markers, including Syn/SYP and CgA¹²⁴. Conversion to NEPC has been associated with genomic alterations common to NE tumors (RB1 loss and TP53 deletion events)^124,126.

n-MYC has emerged as a significant driver of the NEPC phenotype with roles in its initiation and maintenance^124,47 (Figure 5). MYCN gene amplification and overexpression is found in 40% of NEPC tumors²⁰⁹. Unsurprisingly, prostate tumors that express high n-MYC are castration resistant and metastatic in nature. Murine models of NEPC show that n-Myc fuels tumor growth, and these tumors are “addicted” to n-Myc activity, as n-Myc withdrawal rapidly slows tumor proliferation markers, growth, and metastasis¹²⁶.

n-MYC & AKT Functionally Linked in NEPC

n-MYC has important roles in both PAC and NEPC development, as n-MYC is sufficient to transform human prostate epithelial cells and to support lineage plasticity¹²⁶. To achieve this transformation, n-MYC was overexpressed in human prostate epithelial cells along with activated myristoylated AKT Serine/Threonine Kinase 1 (AKT1) (Myr-AKT1). A n-Myc/myrAKT1 expressing single cell clone was sufficient to initiate both PAC and NEPC¹²⁶, supporting a common clone between both PAC/NEPC subtypes and an epithelial to NE transition in PCa. Resulting tumors were castration resistant with low levels of AR expressed and showed NE markers¹²⁶. In the NEPC tumors, n-MYC was highly expressed and tightly correlated with AKT1 levels and activity¹²⁶ (Figure 5). Interestingly, oncogenic serine/threonine kinase AKT1 signaling is known to enhance n-MYC protein stability²¹¹. Molecular experiments showed that inhibiting AKT1 was sufficient to suppress n-MYC protein levels, suggesting that oncogenic n-MYC and AKT1 function are linked and possibly involved in a feed-forward mechanism¹²⁶. It has been suggested that primary PAC subsets with elevated n-MYC acquire stem-like properties and, after treatment, can support growth of NEPC populations¹²⁶.

Emerging Cooperation of n-MYC & AURKA in NEPC

Molecular profiling of NEPC, PCA, and benign prostate tissue identified overexpression and gene amplifications of AURKA, an upstream activator of AKT, and n-MYC in 40% of NEPC and only 5% of PCA¹²³. AURKA functions mostly in mitosis but its ability to complex with n-MYC has been observed in neuroblastoma¹²³. Data supported a cooperation between AURKA and n-MYC in inducing the NE-like state observed in NEPC¹²³. In NEPC, treating tumors with inhibitors targeting AURKA suppressed NE marker levels, n-MYC protein levels, and resulted in tumor regression¹²³. Additional studies have profiled NEPC tumors and found that AURKA and n-MYC are well correlated in patient samples, providing an opportunity to therapeutically target NEPC tumors.

n-MYC Molecular Program & Phenotype in NEPC

Further studies sought to re-affirm n-Myc as an oncogenic driver and clarify the functional role of n-MYC in NEPC. n-MYC overexpression in pre-clinical models drove aggressive PCa recapitulating NEPC²¹². Elevated levels of n-MYC supported NEPC growth and plasticity by reducing cellular dependency on AR signaling and by actively downregulating the transcription of key AR genes. By doing this, NEPC cells remained insensitive to AR inhibitors. In addition, in these cells, high levels of n-MYC actively complexed with the histone methyltransferase, Enhancer of zeste homolog 2 (EZH2) (Figure 5) and AURKA¹²⁴. This novel complex can remodel the chromatin and transcriptional landscape of these cells, fueling uncontrolled growth and tumor evolution. Experiments using murine models and cellular systems showed that this reprogramming sensitizes these cells to both EZH2 and AURKA inhibitors¹²⁴. These findings have been well supported by transcriptional profiling of human NEPC tumors which have similar transcriptional patterns when n-MYC is amplified¹²⁴.

n-MYC amplification likely contributes to the cellular reprogramming of these cells in multiple ways. Molecular profiling of NEPC cells treated with enzalutamide (AR signaling pathway inhibitor) identified high n-MYC and RB1 loss as critical components for E2F1 and CDK5 fueled tumorigenic growth. In this setting, genome integrity is compromised, sensitizing the cells to PARP inhibitors²¹³. Additional studies have also identified alterations in DNA damage efficiency in NEPC cells. In these cells, n-MYC drives contrasting mechanisms regulating the levels of the DNA damage sensing kinase Ataxia Telangiectasia Mutated (ATM). In a subset of NEPC cells, n-MYC-mediated upregulation of miRNA-421 directly suppresses ATM levels. In contrast, the overexpression of n-MYC in different NEPC cell lines increased ATM and resulted in treatment resistance²¹⁴.

2.5. MYC in Neuroblastoma

Neuroblastoma (NB) is a NE tumor that often forms in utero, with the majority of NBs diagnosed before the age of 3²¹⁵. NB is the most common extracranial childhood solid tumor. NB originates from neural crest cells (Figure 6A) that develop from the embryonic ectoderm. These cells then disseminate along the sympathoadrenal track, most often forming at the adrenal medulla, but also occurring at the paraspinal ganglia and abdominal cavity adjacent to the aorta²¹⁶. Due to biological heterogeneity and likely other unknown factors, there is a remarkably diverse clinical course for these tumors, ranging from regression to benignity to aggression leading to death.

Figure 6: Oncogenic n-MYC Promotes Neuroblastoma Tumorigenesis and Vulnerability to Cell Death-Inducing Agents.

(A) Multipotent progenitor cells originating from the neural crest express high levels of n-MYC. These cells migrate and, in unperturbed differentiation, n-MYC is downregulated, allowing progenitors to develop into a collection of autonomic neural cells (right). Aberrant, persistent high n-MYC expression confers stemness while supporting cellular stress and metabolic reprogramming processes. This allows for self-renewal, supporting neuroblastoma development (left). (B) High n-MYC leads to elevated replicative and metabolic cell stress in neuroblastoma, supporting a vulnerability to cell death-inducing agents. In the case of pro-apoptotic BCL-2 member PMAIP1 (NOXA) (left), n-MYC binds to the PMAIP1 promoter, leading to its upregulation and downstream sensitization to the BCL-2 inhibitor, venetoclax. n-MYC also sensitizes neuroblastoma to the iron-dependent cell death ferroptosis. n-MYC increases ROS (driven in part by the increase of intra-cellular iron) and, thus, a reliance on cysteine and glutathione to eliminate this stress. Due to this process, MYCN-amplified neuroblastoma shows sensitivity to ferroptosis inducers, such as GPX4 inhibitors (right). Adapted from:²⁸⁵

While there are several negative prognostic markers for NB, including genomic loss of 1p and 11q, gain of 17q, and the presence of undifferentiated tumoral cells²¹⁷, the status of the MYCN gene remains the most important prognostic marker²¹⁸. High-level amplification of MYCN is the most common reoccurring gene abnormality at diagnosis and following chemoresistance. With few exceptions, MYCN amplifications confer high-risk and are responsible for nearly half of all high-risk NB tumors. Non-genomic-induced high levels of c-MYC, found mutually exclusively of MYCN amplification, is noted in up to 10% of high-risk NB tumors²¹⁹. Thus, like other NE tumors, MYC paralogs play central roles in tumorigenesis.

Understanding how n-MYC impacts the biology of NB tumors has been a long-standing and intense field of investigation²²⁰ (Figure 6A). The importance of this is further evident since n-MYC itself remains undruggable, therefore identifying the important pathways n-MYC regulates may convert to druggable targets. In addition to downstream n-MYC targets, understanding how n-MYC is regulated may help identify upstream druggable targets. A recent study²²¹ screening for kinase regulators of n-MYC demonstrated that PI3K, CK2, CHK1 and STK38 all contribute to the protein stability of n-MYC. AURKA has also be shown to bind directly to n-MYC, preventing its proteasomal-mediated degradation²²². Alisertib, an AURKA inhibitor, induces a conformational change in AURKA that blocks this interaction with n-MYC²²². However, clinical trials of alisertib, in combination with irinotecan and temozolomide, did not yield superior responses in patients with MYCN amplified NB tumors compared to patients with MYCN un-amplified NB tumors²²³. Other therapeutic strategies like proteolysis-targeting chimeras (PROTACs) that degrade AURKA may be more promising²²⁴.

2.5.1. MYCN and Cell Death in Neuroblastoma—An Exploitable Achilles’ Heel?

An intriguing aspect of n-MYC in NB is it induces a paradoxical sensitization to apoptosis-inducing therapies^153,225,226 (Figure 6B). The simplest explanation is that elevated n-MYC induces high levels of replicative and metabolic stress on cells, sensitizing them to apoptosis-inducing drugs by lowering the apoptotic threshold. In addition, high n-MYC levels leads to E-box binding promiscuity, where n-MYC will bind aberrantly and induce the transcription of genes that contain non-canonical E-boxes. One such example is the pro-apoptotic BCL-2 member PMAIP1 (NOXA), where n-MYC binds to the PMAIP1 promoter and upregulates it²²⁵ (Figure 6B), thereby sensitizing cells to the BCL-2 inhibitor venetoclax^225,227,228. Combination therapies of venetoclax and etoposide or cyclophosphamide in four refractory/relapse NB metastatic patients who were heavily pretreated – all achieved stable disease. Interestingly, 1 of the 2 patients with MYCN amplification remained with stable disease 18 months later during the last evaluation²²⁹. While this represents a small number of patients, this is encouraging in such a heavily pre-treated population. Ultimately, venetoclax could serve as the backbone of rational combinations with other targeted therapies for marked responses^225,227, where venetoclax has excelled in this role in hematological cancers^230–233. For instance, combining venetoclax with alisertib led to enhanced cell death and tumor regressions in mice²²⁵. Similarly, combining venetoclax with MDM2 inhibitors, which have demonstrated tolerability and activity in acute myeloid leukemia (AML)²³³, is synergistic in MYCN-amplified NB²²⁷.

2.5.2. n-MYC and Ferroptosis in Neuroblastoma

Ferroptosis is a relatively newly described form of iron-dependent cell death that is caused by the accumulation of iron-dependent reactive oxygen species (ROS) resulting in lipid peroxidation-mediated cell death²³⁴. Ferroptosis is primarily combated in cells by the glutathione-utilizing, phospholipid hydroperoxidase, GPX4 (Figure 6B). Increasingly, ferroptosis inducers (FIs), such as glutathione or GPX4 inhibitors, are being recognized as having the potential of being a new class of anti-cancer drugs²³⁵. Which cancers and how different oncogenes and tumor suppressors impact the sensitivity to FIs is currently a topic of much interest.

In addition to sensitizing to apoptosis inducers like venetoclax, we^236,237 and others^238,239 have recently demonstrated n-MYC sensitizes neuroblastoma to an iron-dependent cell death, ferroptosis (Figure 6B). Indeed, RNAi screens in DepMap revealed MYCN-amplified NB are the most sensitive to GPX4 genetic targeting²³⁶. This therapeutic vulnerability is in part through n-MYC-induced increases in reactive oxygen species (ROS) and subsequent reliance on cysteine and glutathione to eliminate ROS^236–238 in which the increase of extracellular iron (to drive its proliferation program) is at least in part responsible^236,239. As a result, MYCN-amplified NB are exquisitely sensitive to a number of FIs, including GPX4 inhibitors, system Xc inhibitors, and GCLC inhibitors^236–239.

Overall, n-MYC induces cell stress and ROS in NB, resulting in a vulnerability to cell death-inducing agents. These drugs may be important additions to combat this deadly pediatric cancer.

2.6. MYC in Pheochromocytomas & Paragangliomas

Pheochromocytomas and Paragangliomas (PPGLs) are rare NE tumors that develop in the adrenal glands and in the pelvis, chest, neck, and head of patients. Tumor characteristics and patient prognosis varies greatly. PPGL tumors are driven by an array of different genomic aberrations that fuel changes in the DNA damage response, proliferation, apoptosis, and differentiation pathways²⁴⁰. PPGL tumors that metastasize diminish survival times for patients and are associated with microsatellite instability (MSI) and higher mutational burdens²⁴⁰. Although, aberrations in MYC are not found in PPGL tumors, a subset of PPGL patients have mutations within the MYC’s binding partner, MAX^241,242.

MYC-associated factor X, MAX, heterodimerizes with MYC family members and functions to promote MYC transcriptional activity. Tumor sequencing efforts have identified MAX mutations in a number of tumor types that originate from the neural crest, including PPGL. MAX mutations are found in ~2% in PPGL tumors^240,242 with truncating and missense mutations occurring predominantly within exon 3 and 4²⁴¹. These alterations fall within important bHLH-Zip regions²⁴¹ and likely significantly diminish MAX function. The effect of these mutations on the transcriptome, treatment resistance, and outcome for PPGL patients remain untested. However, based on the other malignancies with disruptions in MAX function, it is likely that these tumors have altered MYC activity and genome occupancy.

Although amplification of MYC has not been identified in PPGL tumors, MYC has important and well-defined roles in regulating neural crest development. As these tumors are derived from this tissue, MYC activity is likely already high and key for fueling the expression of a number of PPGL markers, including Cyclin Dependent Kinase1 (CDK1) and Marker of Proliferation Ki67 (Ki67)²⁴⁰. In agreement, the expression of the anti-apoptotic protein, BCL2, correlates with c-MYC levels in PPGL²⁴³. The role of c-MYC and MAX in modulating the transcriptional environment of PPGL to support tumorigenesis, treatment resistance, and metastasis remains unexplored, however growing evidence suggests that c-MYC/MAX are central for PPGL.

3. Targeting the MYC Family

3.1. “Undruggable” MYC

Despite the strong oncogenic potential of MYC and its diverse roles in tumorigenesis, the MYC transcription factor family remains largely “undruggable” or difficult to target pharmacologically²⁴⁴. Historically, targeting MYC has remained difficult due to the nature of the transcription factor family as well as its molecular and structural complexities.

An often-cited concern is that the MYC transcription factor family promotes growth signals in both normal proliferating cells as well as tumor cells. The family is expressed ubiquitously, becoming activated during embryogenesis and in highly proliferative tissues, e.g. gut and skin epidermis^245,246. Direct MYC inhibition may cause deleterious toxicity in normal tissues due to the multifunctional roles of the MYC family in most signaling pathways of most cell types²⁴⁷. As discussed earlier in this review, constitutive knockout MYC (c-MYC^−/−) mice die by embryonic day 10.5 with defects in growth, cardiac and neural development, and failure in vasculogenesis and erythropoiesis^112,248. Importantly, these phenotypes were not observed in an adult preclinical mouse model of Ras-induced lung adenocarcinoma in which endogenous MYC function was reversibly inhibited by the inducible expression of the dominant-interfering MYC mutant Omomyc²⁴⁹.

The MYC family presents significant challenges for drug development due to its conserved structure. Impeding targeted and structure-guided drug development, MYC lacks an enzymatic pocket for small molecule inhibition, exhibits intrinsically disordered functional domains among its protein structure, and has large protein-protein interaction interfaces^115,244,245. It also predominantly shows nuclear localization, prohibiting antibody access for therapeutic intervention^245,250. In addition, the redundancy of the MYC family members and the MYC/MAX high affinity interaction have collectively hindered the design process of effective MYC inhibitors¹¹⁵.

3.2. How can we target MYC?

Recognizing these significant molecular and structural roadblocks, attempts have been made to inhibit MYC directly and indirectly through several strategies.

3.2.1. Direct MYC Inhibition

Small Molecule Inhibitors

Despite the inherent difficulty of inhibiting MYC, there have been several iterations of small molecule inhibitors developed to directly target MYC. A large scale in silico screen coupled with in vivo and in vitro analyses lead to the identification of a small molecule inhibitor MYCi361. MYCi361 showed efficacy in vivo, and could suppress tumor growth, increase immune cell infiltration, upregulate PD-L1, and sensitize tumors to anti-PD-1 immunotherapy. MYCi361 reported a narrow therapeutic index in acute toxicity studies, leading to the consequent development of an improved, more tolerable analog, MYCi975 (Figure 7)^115,251.

Figure 7: Targeting MYC.

MYC is difficult to target and often deemed “undruggable” among literature. Despite this, therapies have emerged to mitigate the strong oncogenic potential of MYC. Therapeutic approaches can be divided based on how they inhibit MYC and its activities: direct inhibitors (left) vs. indirect inhibitors (right) that block or degrade MYC interacting proteins. (Left) Small molecule inhibitors (i.e. MYCi975) have been developed to directly target MYC. MYCi975 destabilizes MYC by interacting with its HLH domain, affecting MYC phosphorylation. Mutant protein Omomyc (left) directly inhibits MYC by heterodimerizing with MYC’s binding partner, MAX, actively outcompeting MYC at E-box binding sites at MYC target genes. (Right) Repressing transcription of MYC, BRD4 inhibition by small molecule BET inhibitors (i.e. JQ1) prevents BRD4’s histone interactions, maintaining chromatin compaction. While PROTACs (right), including ARV-771/ARV-825, are able to degrade BET proteins, leading to MYC inhibition. Aurora Kinase inhibition (right) effectively disrupts and destabilizes the n-MYC/Aurora A protein complex, resulting in MYC ubiquitin-mediated proteolytic degradation and disrupted n-MYC growth signaling. CHK1 inhibition (right) prevents efficient DNA damage repair in malignancies with elevated MYC, promoting genomic instability and apoptosis. Adapted from:²⁸⁶

MYCi975 exhibited anti-tumor efficacy in vivo and promising pharmacokinetic profiles. MYCi975 binds to c-MYC’s HLH domain directly, supporting T58 phosphorylation and consequent proteasome-mediated degradation of MYC. This destabilizes c-MYC, impeding its transcriptional activity and inhibiting MYC-dependent cancer cell viability. In vivo, MYCi975 induced immunogenic cell death, allowing increased T-cell infiltration and ultimately the upregulation of PD-L1 in the TME. In FVB mice bearing Myc-CaP (MYC-driven prostate cancer cell line) tumors, previously established to be resistant to anti-PD-1 immunotherapy²⁵², MYCi975 suppressed tumor growth and showed synergy when combined with anti-PD-1 immunotherapy. Through sensitizing refractory tumors to the immune checkpoint blockade, this combinatorial approach supports the emerging idea to combine the immune checkpoint blockade with MYC inhibition in MYC-associated malignancies^115,251.

Omomyc

Another strategy for directly inhibiting the MYC family involves modifying the selectivity of the bHLHZip protein. Identification of MYC’s protein-protein recognition sequence within the leucine zipper enabled the development of the dominant negative Omomyc protein²⁵³ (Figure 7), which is capable of homodimerizing and/or heterodimerizing with MYC or MAX. ChIP and Proximity Ligation assays suggest that Omomyc preferentially interacts with MAX and can outcompete MYC for E-box binding on MYC target genes, diminishing MYC fueled transcription^254,255. Omomyc has some pharmacokinetic limitations in vivo, rapidly distributing to the kidneys and liver with a short half-life in plasma²⁵⁴. Recognizing the potential of MYC targeted therapeutic intervention in a MYC-associated NE disease, Omomyc suppressed cell growth in a large panel of SCLC cell lines through G1 cell cycle arrest and apoptosis²⁵⁶. This effect is dependent on CDKN1A activation and the TP73 pathway²⁵⁶.

In 2021, Omomyc (OMO-103) began Phase 1 and 2 clinical dose-escalation trials (NCT04808362/MYCure), becoming the first direct MYC inhibitor to reach clinical trial. OMO-103 suggested Omomyc is safe and reported common low-grade infusion-related reactions, e.g. chills, rash, hypotension. The inhibitor reported suitable pharmacokinetics up to the recommended dose, showing little to no immunogenicity. However, the study is limited by the omittance of MYC amplification or expression in the patient inclusion criteria. Future directions must address whether MYC expression level might affect OMO-103 sensitivity^115,257.

3.2.2. Indirect MYC Inhibition

Research has largely focused on indirectly inhibiting MYC through targeting MYC interacting proteins. To encompass this vast field of study, we have highlighted 4 key proteins (BRD4, AURKA/B, ATR, CHK1) being targeted for inhibition and a relatively new approach (PROTACs) to mitigate MYC’s activity (Figure 7).

BRD4 Inhibition

BRD4, a chromatin binding protein in the Bromodomain and Extra Terminal (BET) family, functions in transcriptional and epigenetic regulation²⁵⁸. BRD4 acts as a histone acetyltransferase (HAT), acetylating both histones H3 and H4. Notably, BRD4 acetylates H3 at K122, a residue vital to nucleosome stability. Acetylation of K122 promotes nucleosome clearance, chromatin decompaction, and provides access for transcriptional machinery, including MYC⁶⁴. Recognizing the interplay between MYC transcription and BRD4, small molecule BET inhibitors have emerged²⁵⁹. Namely, JQ1, a small molecule BRD2/4 inhibitor, is known to engage with BRD2/4’s bromodomain pockets, limiting potentiating interactions with acetylated histones. This inhibition effectively represses the transcription of MYC and, consequently, MYC target genes^{62,115,259,260} (Figure 7). Improving BET inhibitors’ pharmacokinetics, several iterations of BET inhibitors have followed but remain in Phase 1 and 2 clinical trials (i.e. AZD5153, BMS-986158, CPI-0610, GSK525762, INCB057643, FT-1101, CC-90010, ODM-207). Among these trials, all BET inhibitors reported exposure-dependent thrombocytopenia limiting their continued clinical application. Future work must clarify the optimal dosing schedule and therapeutic combinations to support the clinical use of BET inhibitors^115,261.

PROTACs

The approach of using PROTACs (PROteolysis-TArgeting Chimeras) to degrade MYC partners (Figure 7) has shown moderate success in pre-clinical models. PROTACs selectively degrade targeted proteins by taking advantage of the ubiquitin proteasome degradation system²⁶². Beneficial for MYC-driven malignancies, PROTACs are uniquely able to target proteins for degradation without interacting with an active enzyme site. Showing advantages over conventional small molecule inhibitors, PROTACs work at a lower dose and function catalytically. PROTACs mechanistically form a ternary complex with an E3 ligase, and the desired target protein meant for degradation²⁶³.

PROTAC ARV-771 was able to degrade BET proteins in CRPC, and consequently, diminished c-MYC expression^115,264. Whereas BET inhibitor and PROTAC ARV-825 suppressed proliferation in NB cells while promoting cell cycle arrest and apoptosis. ARV-825 reduced BET protein expression, consequentially diminishing c-MYC and n-MYC expression. Further potentiating ARV-825 for therapeutic intervention in NB, ARV-825 reduced tumor growth and downregulated n-MYC and BRD4 in NB xenografts^115,265.

Although PROTACs against MYC partners remain largely pre-clinical, one PROTAC has begun to show promise in clinical trials in heavily pretreated CRPC. Bavdegalutamide (ARV-110), an oral PROTAC protein degrader that targets the androgen receptor (AR), showed partial responses (tumor reduction) in Phase 1 and 2 clinical trials (NCT0388861) in post NHA, heavily pretreated metastatic CRPC. Enhanced activity was observed in patients with AR T878X and/or H875Y mutations, indicating the presence of a ARV-110 sensitive population among patients with pretreated metastatic CRPC. Further investigation into the effectiveness of ARV-110 in this biomarker-defined subgroup is warranted^266,267.

Aurora Kinase Inhibition

Inhibition of Aurora kinases (AURKA/B) have been of interest in MYC-associated malignancies, especially in the case of n-MYC and NB. Aurora A kinase stabilizes n-MYC by sequestering it and interacting with SCF^Fbxw7 ubiquitin ligase, saving it from ubiquitin-mediated proteolytic degradation^268,269. Numerous inhibitors targeting Aurora kinases have been developed that are largely selective for AURKA (i.e. alisertib, LY3295668), AURKB (i.e. AZD1152), or both Aurora kinases (i.e. SNS-314, danusertib)^270,271. A common mechanism of action of these inhibitors can be described through alisertib. Inhibitor alisertib disrupts and destabilizes (Figure 7) the n-Myc-Aurora A protein complex, prohibiting n-MYC signaling and mediated growth²⁷².

Interestingly, in a Phase 2 clinical trial studying alisertib/paclitaxel compared to placebo/paclitaxel in SCLC, c-MYC expression or RB1, RBL1, RBL2, CDK6 mutations correlated with improved progression free survival (PFS) and overall survival (OS) in the alisertib group²⁷³. In addition, alisertib is currently being tested in another Phase 2 clinical trial (PUMA-ALI-4201: NCT06095505). The trial is evaluating the use of alisertib as a monotherapy in SCLC patients currently on or that have received platinum-based chemotherapy and anti-PD-L1 immunotherapy. This study is ongoing and aims to identify the biomarker subgroup that will most benefit from alisertib while further clarifying the efficacy, pharmacokinetics, and safety of alisertib (https://www.cancer.gov/research/participate/clinical-trials-search/v?id=NCI-2023-10166&r=1). Pre-clinical rationale for this study further supports that models with high c-MYC expression and/or RB1 loss of function exhibit the greatest alisertib sensitivity.

ATR/CHK1/WEE1 Inhibition

When single-stranded DNA is generated at DNA damage sites or stressed replication forks, RPA-coated single-stranded DNA activates ATR and its binding partner ATRIP. ATR consequently phosphorylates its downstream effector CHK1 at Ser-317 and Ser-345. Activated CHK1 promotes the intra-S and G2/M checkpoints, promoting cell cycle arrest for appropriate DNA damage repair^274,275.

Interesting to NE malignancies, if both TP53 and RB1 are lost, CHK1 also becomes upregulated, further building a dependency on the ATR/CHK1 signaling pathway to active appropriate cell cycle arrest for DNA damage repair²⁷⁵. Cancers with NE features may share a particular therapeutic vulnerability to inhibition of proteins involved in DNA damage repair, such as PARP, cell cycle checkpoints (ATR/CHK1), and DNA damaging agents (platinum chemotherapies/topoisomerase (TOP1) inhibitors)¹⁸². This is likely due to high rates of replication stress and DNA damage caused by RB1-loss and/or MYC amplification.

Cancers with elevated MYC have been suggested to show increased vulnerability to CHK1 inhibition (Figure 7). The mechanism of how c-MYC regulates CHK1 is unclear and warrants further investigation⁶. It has been hypothesized that cancers with TP53 mutations and elevated MYC levels become dependent on CHK1 to maintain their genomic integrity^276,277. In these cells, MYC overexpression increases CHK1 expression, independently of the DNA damage response proteins, ATM and p53^6,276. This is particularly clear in SCLC cell lines, where elevated MYC mRNA correlated with high CHK1 protein expression and greater DNA repair capacity⁶. In this setting, CHK1 inhibition prevents efficient DNA damage repair in malignancies with elevated MYC, resulting in genome instability and apoptosis⁶.

In a Phase II proof-of-concept clinical trial²⁷⁸, the ATR inhibitor, berzosertib (M6620), was combined with the TOP1 inhibitor topotecan in patients with relapsed small cell neuroendocrine cancers (SCNCs). SCNCs can develop in many sites, including commonly the lung (SCLC). SCNCs can occur de novo or via mechanisms involving cellular plasticity and suppression of lineage defining factors. Interestingly, this study found that responding tumors shared a notable enrichment of high NE differentiation (all were high NE subtypes ASCL1 or NEUROD1) and heightened replication stress. This therapeutic combination resulted in tumor regression, even in heavily pretreated patients with platinum-resistant disease²⁷⁸. Currently, another Phase 2 clinical trial (NCT03896503) is active in SCLC following this combination approach and includes a cohort for patients with extra-pulmonary-SCNC²⁷⁸.

CHK1 can also mediate G2/M cell cycle arrest through phosphorylating CDC25B at Ser-323 and CDC25C at Ser-216. This results in their degradation and suppresses the activation of CDK1/cyclin B kinases that control the G2/M checkpoint²⁷⁴. WEE1 further promotes G2 arrest to allow for DNA repair before entering mitosis by inhibiting CDC2/CDK1 activity through phosphorylating CDK1. Inhibition of CHK1 and/or WEE1 increases replication stress and genomic instability, effectively removing the G2/M cell cycle checkpoint and forcing premature mitosis with unrepaired DNA. This can ultimately result in mitotic catastrophe and apoptosis²⁷⁵.

Following this rationale, the CHK1 inhibitor, SRA737, was combined with WEE1 inhibitor AZD1775 in metastatic CRPC. These inhibitors showed strong synergistic effects. Both agents, in combination or as monotherapy, were also able to diminish the tumor growth and metastasis of NEPC tumors in a TRAMP genetically engineered transgenic prostate cancer murine model. Given in a 2-way sequential combination treatment, AZD1775 and SRA737 showed higher efficacy in combination than each monotherapy. Exploring the mechanism, SRA737 prevented AZD1775-induced feedback activation of CHK1. This consequently promoted mitotic entry and led to the accumulation of DNA damage. Supporting the potential of CHK1 inhibition in NE cancers, SRA737 showed high efficacy as a monotherapy, blocking NEPC proliferation and metastasis while also prolonging survival in the TRAMP murine model. Overall, this study reinforces the emerging value of CHK1 and WEE1 inhibition in CRPC and largely NE tumors²⁷⁵.

4. Outstanding Questions and Future Directions

4.1. Why is there a parallel between MYC’s roles in varying NE cancers?

Resistance-Plasticity-Cell Fate?

NE transformation has emerged as a powerful mechanism of acquired resistance. To exemplify this, consider the parallels among lung and prostate adenocarcinomas (Figure 8). Both malignancies are initially sensitive to their corresponding treatments: prostate adenocarcinomas respond well to antiandrogen therapy^279,280 and EGFR-mutant lung adenocarcinomas are sensitive to EGFR tyrosine kinase inhibitors (TKIs)^280,281. Despite promising initial responses, most patients develop acquired resistance to these suppressive agents^279–281 (Figure 8). Likely, EGFR and AR signaling, in the lung and prostate respectively, oppose NE transformation, supporting an epithelial lineage. One hypothesis in the field suggests that the suppression of these signals by targeted treatment enables NE transformation²⁸⁰ and resistance.

Figure 8: Parallels of Neuroendocrine Differentiation & MYC Family.

Strong similarities can be drawn from the epithelial to neuroendocrine-like state transition observed in lung, prostate, and pancreatic ductal adenocarcinomas. Following TP53 loss, a driving mutation (i.e. EGFR or KRAS), applied therapeutic pressure, and usually RB1 loss, these malignancies acquire drug resistance, along with a marked upregulation of a MYC family member. These MYC family members have been implicated in supporting lineage plasticity and conferring intratumoral heterogeneity. Adapted from:²⁸⁰

The nearly concurrent inactivation or loss of TP53 and RB1 remains an early event in NE transformation, no matter the site of the tumor, supporting plasticity and lineage switching²⁸². Most tumors with altered TP53 and RB1 do not complete the full NE terminal differentiation resulting in transformation²⁸³. Notably, in this setting, TP53 and RB1 loss did not induce NE transformation, but were sufficient for acquired resistance to EGFR inhibitors²⁰¹. Additional oncogenic signaling or factors, including MYC, BCL-2, SOX, or AKT (Figure 8) are needed to transform into a more NE-like phenotype from lung and prostate adenocarcinomas^280,284.

Suppressive, inhibitory treatments when combined with oncogenic signaling like those of MYC family members have been implicated in cellular plasticity and may provide a platform for acquired resistance²⁸⁰ (Figure 8). Further work is required to tease out MYC’s complicated mechanistic role in this NE transition, but it is known to support resistance and lineage plasticity. In epithelial prostate cells of Pten KO mice, n-MYC overexpression results in antiandrogen resistance and a higher incidence of NE tumors^{123,124,209,280}. Additionally, in Kras-mutant pancreatic cancer murine models, c-MYC overexpression selected for a NE phenotype in a tumor cell subpopulation resistant to gemcitabine¹²⁷ (Figure 8).

4.2. How is MYC contributing to NE tumorigenesis and resistance?

Despite the global effects of MYC on proliferation, metabolism, cell cycle progression, and apoptosis, little work has been done to molecularly characterize the role of MYC in NE tumorigenesis and drug resistance. Global mapping of the MYC binding sites is required to understand its transformative effects in NE cancers. Building on this, temporal studies are also needed to elucidate the vital, cryptic roles of the MYC transcription factor family during NE drug resistance. This future avenue of experiments will be critical for understanding what essential proteins and pathways MYC is modulating in NE cancers.

4.3. What’s next?

Unfortunately, drugging known MYC-modulated pathways or proteins that interact with or regulate MYC, like Aurora A, have not yet succeeded in clinical trials. While progress has been made to directly drug the MYC family members, the drug properties of these compounds have limited clinical appeal. PROTACs and more drug-friendly small molecule inhibitors are now actively being developed across academia and industry to address this need. Vitally, it is necessary to investigate the signaling upstream of MYC, identify its key regulators, and to tease out how MYC is modulating the transcriptional landscape, all with the hope of finding a druggable candidate for NE tumors.