Targeting low-risk myelodysplastic syndrome with novel therapeutic strategies

Abstract

Myelodysplastic syndrome (MDS) is a group of hematopoietic disorders with limited treatment options. Anemia is a common symptom in MDS, and although erythropoiesis-stimulating agents such as erythropoietin, lenalidomide, and luspatercept are available to treat anemia, many MDS patients do not respond to these first-line therapies. Therefore, alternative drug development strategies are needed to improve therapeutic efficacy. Splicing modulators to correct splicing-related defects have shown promising results in clinical trials. Targeting differentiation of early erythroid progenitors to increase the erythroid output in MDS is another novel approach, which has shown encouraging results at the pre-clinical stage. Together, these therapeutic strategies provide new avenues to target MDS symptoms untreatable previously.

MDS: prevalence and classification

MDS is characterized by multilineage cytopenias arising from ineffective hematopoiesis, increased apoptosis of diseased cells, and disruption of hematopoietic cell differentiation (Figure 1A) [1–4]. More than 10000 new cases of MDS are reported every year in the United States and close to 87000 worldwide. There are major clinical consequences associated with MDS that lead to morbidities, mainly caused by cytopenias and the potential for MDS to evolve into acute myeloid leukemia (AML) [5–7]. Progression to AML is mostly associated with high-risk MDS (HR-MDS) which accounts for 30% of the total MDS patients with survival <2 years [1, 8]. The early MDS initiating mutations at stem cell level together with secondary acquired mutations associated with AML give rise to leukemic stem cells that cause the transformation of MDS to AML [5]. As ~75% of people affected by MDS are >60 years old, these patients cannot tolerate intensive therapeutic approaches such as chemotherapies or benefit from allogeneic hematopoietic stem cell transplantation (allo-HSCT) (see Glossary), and often these treatments are too harsh for elderly patients [9–11]. Therefore, treatments for MDS are risk-adapted, involving the definition of different therapy goals according to the patient’s risk status [9, 12].

Figure 1. MDS and current therapeutic options.

A) Schematic diagram depicting the peripheral blood of normal individuals and MDS patients. Peripheral blood of normal individuals shows balanced hematopoietic output, whereas peripheral blood of MDS patients exhibits reduced blood cell production. B) Several treatments are available for low-risk MDS patients. Erythropoietin (EPO) works through the Janus kinase/signal transducer and activation of transcription (JAK-STAT) pathway to induce hematopoietic gene transcription. The Del (5q) specific drug lenalidomide acts by ubiquitin (Ub)-mediated degradation of specific substrates thereby inducing apoptosis, and also by inhibiting the cell cycle specific gene, cell division cycle 25C (CDC25C), to arrest Del (5q) MDS cells in G2/M phase. Luspatercept contains a modified version of the extracellular domain of the activin receptor IIB (ActRIIB), which inhibits transforming growth factor (TGF-β) signaling and blocks abnormally elevated SMAD signaling, to promote erythroid cell maturation. Hypomethylating agents (HMAs) are agents of choice for high-risk patients. HMAs inhibit DNA methyltransferase (DNMT) to ameliorate increased CpG island methylation, that is characteristic of abnormal hematopoietic cells in MDS, and exert cytotoxic effects.

The International Prognostic Scoring System (IPSS) has been an essential standard for assessing the prognosis of MDS patients, along with a comprehensive diagnostic workup and testing for molecular abnormalities [13]. A revised version of IPSS (IPSS-R) is being used in clinics nowadays. IPSS-R uses criteria including the percentage of abnormal blasts in bone marrow, cytogenetics and detailed blood cell parameters to calculate risk scores which provide important prognostic and predictive information for a subsequent response to a given therapy [8]. Patients are classified based on IPSS-R risk score into low-risk MDS (LR-MDS) and high-risk MDS (HR-MDS) populations. LR-MDS patients exhibit anemia-like symptoms; therefore, the first lines of treatment are erythropoiesis-stimulating agents (ESAs) and red blood cell (RBC) transfusion. HR-MDS patients exhibit AML-like symptoms and are offered more intensive treatments, including chemotherapy with hypomethylating agents (HMAs) and allo-HSCT [14].

Constantly evolving and advancing technologies, particularly bulk and single-cell sequencing methodologies, have improved our understanding of MDS by pinpointing somatic mutations and their roles in the pathogenesis of the disease [15–18]. Several studies examining large numbers of MDS samples have identified ~40 recurrently mutated genes in various categories with almost all of the patients, including those with normal cytogenetics [19], harboring at least one mutation [19, 20]. The most frequently mutated genes are splicing factors, epigenetic regulators, and transcription factors [15, 17, 18, 21–28]. This mutational information enables clinicians potentially to tailor a therapeutic regimen for each patient, ultimately giving a better outcome. This review specifically addresses LR-MDS treatments. We present currently available options, and discuss newly advancing therapeutic strategies for this patient group.

Current LR-MDS treatments

Erythropoiesis-stimulating agents (ESAs)

ESAs work through the erythropoietin (EPO) receptor (EpoR), a cell surface receptor that belongs to the cytokine receptor family [29, 30]. Structural studies of this receptor have revealed that it lacks a cytoplasmic kinase domain and, relies on Janus kinase 2 (JAK2) protein tyrosine kinase to exert Epo-responsive signal transduction [31, 32]. Upon activation of EpoR, JAK2 tyrosine kinase residues are phosphorylated and serve as docking sites for intracellular signaling molecules. Phosphorylated JAK2 directly interacts with signal transducer and activation of transcription 5 (STAT5), which serves as a scaffold to induce EpoR signal transduction. STAT5A and STAT5B are predominant signal transducers of EpoR which upon activation, accumulate in the nucleus to induce the transcription of EpoR responsive genes (Figure 1B) [33, 34].

Treatment for LR-MDS patients without RBC transfusion-dependence (less than 2 RBC units per month) often starts with ESAs, such as recombinant EPO or darbepoetin (DAR, which is re-engineered from EPO to increase molecular mass for greater half-life and potency), as the standard of care [35–37]. Of note, MDS patients differ in their serum EPO levels and that determines the response to ESA treatment. Patients with <200 IU/L EPO level are more likely to respond whereas patients with >500 IU/L serum EPO level do not qualify to receive ESA treatment [38, 39]. ESA has been a first-line option in the LR-MDS patients with symptomatic anemia or low RBC transfusion burden. However, most MDS patients already have excessive EPO levels, which correlates with a lack of response to ESAs.

Patients who are non-responsive to ESAs and have <10 g/dL hemoglobin (Hb) concentration are recommended for RBC transfusion. The dose of RBCs and frequency of transfusion is determined by the severity of anemia and varies case by case [40]. In some cases, management of anemia together with RBC transfusion and ESAs helps patients to achieve transfusion independence (TI) [36, 41]. However, frequent RBC transfusions come with their own set of problems. Notable among these, is iron overload toxicity, which arises as excess iron from each transfusion accumulates in vital organs. Without effective iron clearance, there is a high risk of multiple vital organ failures [42, 43]. Iron chelation therapy (ICT) is often used to treat iron overload in LR-MDS patients but has received mixed response in clinics. The primary reason is that the majority of the MDS patients are elderly with other complications, which confounds the safety and tolerance of this therapy. However, there are published research studies and a clinical trial (NCT00940602ⁱ) which suggest that ICT can improve event free and overall survival of low-risk MDS patients and it should be seriously considered for these patients [44, 45].

Lenalidomide

A subset of LR-MDS patients harbor a deletion (Del (5q)) in the long (q) arm of chromosome 5. The commonly deleted region (CDR) known as 5q31 and 5q33 [46], is the location of many MDS-related genes including a core component gene of the 40S ribosomal subunit, ribosomal protein S14 (RPS14) [46–50], cell division cycle 25C (CDC25C) [49, 50], early growth response 1 (EGR1), and several critical micro-RNAs (miRNA) associated with elevated innate immune signaling such as miR-145 and miR-146a [51]. Del (5q) MDS is characterized by distinct cytomorphological abnormalities [52]. It shows relatively high sensitivity to lenalidomide, which exerts its effects through multiple mechanisms. Lenalidomide inhibits the proliferation of MDS cells by targeting cell division cycle 25C (CDC25C) and protein phosphatase 2A (PP2A), which in turn leads to G2/M cell cycle arrest and apoptosis [53, 54]. It has also been reported to bind to cereblon (CRBN), an adapter for the E3 ubiquitin ligase CRL4^CRBN; modulation of the activity of CRL4^CRBN leads to the degradation of disease-specific substrates. An increase in ubiquitination of casein kinase1 alpha (CK1α) and ikaros family zinc finger1 (IKZF1) followed by proteasome-mediated degradation leads to an increase in apoptosis in MDS cells with Del (5q) (Figure 1B) [55, 56].

Del (5q) LR-MDS patients often present with high EPO levels and become RBC transfusion-dependent in their disease course [57]. In multiple clinical trials, lenalidomide treatment has led to hematological improvements (HI) and even transfusion independence in Del (5q) MDS patients [41, 58, 59]. In a phase-3 trial (NCT00179621ⁱⁱ), lenalidomide treatment led to TI for ≥26 weeks in 56.1% of the Del (5q) MDS patients and helped to achieve complete cytogenetic response, clearance of chromosomal abnormalities, in 50% of the patients. Moreover, lenalidomide treatment improved overall survival (56.5%) and lowered the risk of AML transformation (25.1%).

Novel LR-MDS therapeutic agents and strategies

Activin receptor IIB ligand trap - Luspatercept

Luspatercept is a recombinant fusion protein which acts via the transforming growth factor-beta (TGF-β)/SMAD signaling pathway. TGF-β signaling has a well-established role in erythropoiesis and hematopoietic stem cell function and survival [60], and it has a growth inhibitory effect on erythropoiesis at the erythroblast stage [61]. Luspatercept contains a modified extracellular domain of the human activin receptor type IIB (ActRIIB) linked to a human immunoglobulin G1 (IgG1) [61]. In many pre-clinical studies, it has been shown to bind specifically to endogenous transforming growth factor (TGF)-β superfamily ligands [62, 63]. Upon binding it functions as a trap for TGF-β ligands which causes inhibition of abnormally elevated SMAD2/3 signaling (Figure 1B). This leads to an increase in erythroid cell maturation by promoting differentiation of erythroblasts [61].

RBC transfusion-dependent patients, who do not have 5q deletion and have an EPO level >200 IU/L, are less likely to respond to recombinant EPO treatment and become RBC transfusion-dependent [64, 65]. In those cases, luspatercept has been shown to increase the differentiation of erythroblasts to boost erythroid output [66]. In a phase-3 clinical trial (NCT02631070ⁱⁱⁱ), luspatercept has shown hematological improvement and RBC transfusion independence in 63% of patients who otherwise require 2 units of RBCs every two weeks, although its effect on MDS patients with high EPO levels is limited [62].

Splicing mutations and modulators

Splicing factor mutations are prevalent in MDS patients [67]. Serine/arginine-rich splicing factor 2 (SRSF2), splicing factor 3b subunit 1 (SF3B1), U2 small nuclear RNA auxiliary factor 1 (U2AF1) and zinc finger CCCH-type RNA binding motif and serine/arginine-rich 2 (ZRSR2) are the most frequently mutated splicing factors, and these occur in a mutually exclusive manner [21]. SF3B1, SRSF2, and U2AF1 are subject to heterozygous, change-of-function [68–70] missense mutations affecting specific residues [16, 19, 21]. In contrast, the X chromosome-encoded ZRSR2 is enriched in nonsense and frameshift mutations mainly in male patients, consistent with loss of function [16, 19, 21]. Studies in MDS mouse models have shown that point mutations in splicing factors blocked hematopoiesis and induced apoptosis of hematopoietic stem and progenitor cells (HSPCs) [27, 68, 71, 72]. Each point mutation has a distinct effect on gene expression and splicing. While SRSF2 generally affects cassette exon recognition and SF3B1 alters 3′ splice site recognition, U2AF1 mutation causes exon inclusion and alternative 3′ splice site recognition [68, 71]. Although each mutant protein targets different pre-mRNA splicing events, they produce convergent adverse effects on hematopoiesis, for example, via indirect activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway [72]. On the other hand, impaired minor (U12) intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal [73].

SRSF2 is involved in splicing of pre-mRNAs; SRSF2^P95H mutation alters SRSF2 recognition of its normal exonic splicing enhancer (ESE) and causes mis-splicing of key hematopoietic regulator genes. SRSF2^WT/WT normally binds to CCNG or GGNG consensus sequences with similar affinity, whereas SRSF2^P95H/WT mutation leads to an increase in CCNG binding, which leads to degradation of many essential hematopoietic regulator genes (Figure 2A) such as enhancer of zeste homolog 2 (EZH2) [68]. SF3B1^K700E mutation causes excessive usage of an alternative 3′ splice site and aberrant splicing events; ~50% of murine and human aberrantly spliced genes undergo nonsense-mediated decay (NMD). Mis-splicing and NMD of mitogen-activated protein kinase kinase kinase 7 (MAP3K7) due to SF3B1^K700E mutation leads to hyperactivation of NF-kβ signaling (Figure 2B) [71]. SF3B1^K700E mutations are also commonly found in various cancers. Bromodomain-containing protein 9 (BRD9) - a non-canonical BAF complex subunit is highly mis-spliced and degraded in tumors and restoring BRD9 expression reduces tumor growth in vivo [74]. These tumors can also be selectively targeted by splicing modulators. MDS patients carry U2AF1 splicing factor mutations at S34 and Q157 positions. These mutations lead to exon inclusion splicing events and give rise to alternatively spliced versions of many inflammatory and immunomodulatory genes. MDS patients with U2AF1 mutations express a longer version of interleukin-1 receptor-associated kinase 4 (IRAK4-L) gene. IRAK4-L expression gets assembled with myddosome, a receptor-proximal complex which controls innate immune signal transduction, and hyperactivates NF-κβ signaling [75] (Figure 2C). Although the critical targets of ZRSR2 mutation have not been fully elucidated, leucine zipper like transcription regulator 1 (LZTR1), a regulator of Ras-related guanosine triphosphate hydrolyzing enzymes (GTPases), was identified as NMD events of minor intron-containing mRNAs in ZRSR2-mutated MDS cells [73, 76–78].

Figure 2. Splicing alterations in MDS.

A)SRSF2^P95H mutation in Serine/Arginine-Rich Splicing Factor 2 increases affinity for CCNG consensus sequence, as opposed to CCNG or GGNG. Binding at the exonic splicing enhancer (ESE) leads to mis-splicing of EZH2 mRNA and its degradation. B) SF3B1^K700E mutation in Splicing Factor 3b subunit 1 leads to an increase in alternative 3′ splice site usage, which causes mis-splicing and nonsense mediated decay (NMD) of MAP3K7 gene and, ultimately, hyperactivation of NF-κB signaling. C) Mutation at specific residues in U2 small nuclear RNA auxiliary factor 1, U2AF1, causes cassette exon inclusion and alternative 3′ splice site usage. MDS patients with U2AF1 mutation express IRAK4-L, which leads to activation of innate immune signaling.

EZH2, enhancer of zeste homolog 2; IRAK4-L, interleukin-1 receptor-associated kinase 4-long form; MAP3K7, mitogen activated protein kinase kinase kinase 7.

Following the discovery that natural products and their derivatives, such as pladienolides, FR901464, and spliceostatin A, physically bind to the SF3B complex and inhibit pre-mRNA splicing at an early step in spliceosome assembly [79, 80], pharmacological modulation of splicing has been developed as a potential therapeutic strategy for MDS patients [79–81]. E7107, a pladienolide derivative, was developed as an anti-tumor agent to bind to the SF3B complex to inhibit pre-mRNA splicing [80, 82]. The main challenge is to make these modulators more specific to spliceosome mutant cells and more bioavailable. In recent years, a novel medicinal chemistry approach was successfully used to synthesize small molecules to target mutant spliceosomal machinery, resulting in the discovery of the small chemical compound H3B-8800 (an orally bioavailable analogue of E7107) [83]. H3B-8800 binds to both wild-type and mutant spliceosomes but preferentially kills spliceosomal mutant cells. Sequencing data has shown that treatment with H3B-8800 retains short GC-rich introns which codes for important spliceosomal components. Moreover, spliceosomal mutant cells heavily rely on remaining wild-type spliceosome components and are more prone to the effects of splicing perturbation, providing an important mechanism for specificity towards spliceosomal mutant cells [72, 83]. A Phase 1 clinical trial (NCT02841540^iv) of H3B-8800 completed successfully in 2019 (see Clinician’s Corner). In the clinic, H3B-8800 has exhibited good spliceosomal component binding in a dose-dependent manner, and it has been safe even with prolonged dosing [84]. Ongoing clinical trials will further verify its therapeutic efficacy.

Early erythroid progenitor and CHRM4 antagonist

HSPCs are capable of undergoing differentiation to generate mature hematopoietic cells. In the erythroid lineage, the burst-forming unit erythroid (BFU-E) is the first lineage determinant progenitor cell. BFU-E possesses enormous capacity for differentiation, which can generate thousands of erythrocytes. BFU-E differentiates into a late erythroid progenitor, colony-forming unit erythroid (CFU-E) [85]; CFU-E further differentiates into erythroblasts, which can form mature RBCs [61]. CFU-E expresses EpoR and EPO regulates its survival and differentiation [86]. However, specific regulators of BFU-E differentiation are unclear. Studies with human MDS patients have uncovered significantly lower numbers of BFU-E in bone marrow, compared to healthy individuals [87, 88]. These studies have also reported increased EPO levels in MDS patients due to anemic stress. Similarly, low levels of BFU-E, high levels of EPO and anemia symptoms have been reported in splicing factor mutant MDS mouse models [68, 71]. Together, these findings suggest that insufficient BFU-Es generate significantly fewer CFU-Es, which causes the unresponsiveness of MDS patients to EPO, and ultimately reduced RBC production and anemia. Therefore, targeting BFU-E differentiation could be a beneficial and novel approach in treating MDS.

Recently, the G protein-coupled receptor (GPCR), cholinergic receptor muscarinic 4 (CHRM4) was found to be expressed on early erythroid progenitors and to regulate BFU-E differentiation [89]. Blocking CHRM4 results in activation of cyclic adenosine monophosphate (cAMP) and increased cAMP response element-binding protein (CREB) transcription factor activity. CREB regulates many vital genes responsible for transient maintenance of BFU-E progenitor status to allow more RBCs to be generated from each BFU-E (Figure 3). Both genetic and pharmacological inhibition of CHRM4 with selective antagonists resulted in increased erythrocyte production and corrected anemias in mouse models of MDS, aging, and hemolysis [89]. Importantly, pharmacological inhibition of CHRM4 also rescued BFU-E and reduced abnormal EPO to levels comparable to wild-type mice, indicating that pharmacological inhibition of CHRM4 overcame EPO resistance in MDS [89]. These promising pre-clinical efficacies encourage the ongoing downstream pharmacological development of this novel therapy into clinical trials for MDS.

Figure 3. Therapeutic targeting of early erythroid progenitors in LR-MDS.

A) CHRM4 regulates erythropoiesis through the hematopoietic arc (hematopoarc). Neurotransmitter acetylcholine (ACh) is secreted in the microenvironment to act on CHRM4, a Gi coupled GPCR, on early erythroid progenitors. Pharmacological targeting of CHRM4 with antagonists, in pre-clinical setting, treats anemia of LR-MDS. Inhibition of CHRM4 leads to an increase in cAMP-CREB signaling. In the nucleus, phosphorylated CREB (p-CREB) turns on the expression of key erythroid regulators, such as GATA binding protein 2 and ZFP36 ring finger protein like 2, to transiently maintain early erythroid progenitor status and allow more erythrocytes to be produced. B) Erythroid lineage of hematopoiesis and sites of drug intervention. The hematopoietic stem cell (HSC), under the influence of growth factors such as IL3, SCF and GM-CSF, gives rise to a progenitor cell, burst-forming unit erythroid (BFU-E). BFU-E differentiates, with the aid of growth factors, into a colony-forming unit erythroid (CFU-E). CFU-E, in the presence of EPO hormone, differentiates into various stages of erythroblasts and ultimately gives rise to erythrocytes. CHRM4 inhibitor enhances differentiation of BFU-Es independently of EPO, whereas luspatercept increases differentiation of erythroblasts, to increase the production of erythrocytes.

cAMP, cyclic adenosine monophosphate; CHRM4, Cholinergic receptor muscarinic 4; CREB, cAMP response element-binding protein; EPO, erythropoietin; GM-CSF, granulocyte-macrophage colony-stimulating factor; GPCR, G-protein coupled receptor; IL3, interleukin 3; SCF, stem cell factor.

Hematopoietic organs including spleen and bone marrow are highly innervated by cholinergic nerves and nerve terminals which relay stimuli by means of neurotransmitters [90–92]. The identification of a functionally important neurotransmitter receptor CHRM4 involved in regulating BFU-E differentiation is the first example of how the nervous system can directly modulate HSPC differentiation and self-renewal [89]. We coined the term “Hematopoietic Arc (HematopoArc)” for this novel means of regulating hematopoiesis [89]. Further research dissecting each component of this regulation will provide important mechanistic insights.

Concluding remarks

Anemia and anemia-related symptoms are the first signs of MDS, and therefore, treatments with ESAs are the first choice of therapeutics. Conventionally, recombinant erythropoietin and its analogs are used. However, LR-MDS patients often have high endogenous EPO and, thus, they do not qualify to receive these ESAs. In these cases, the recent FDA approved drugs luspatercept and lenalidomide, if patients have Del (5q), could be the drug of choice. Although these agents drive the differentiation of erythroid cells and produce more mature RBCs to boost erythroid output, 80% of patients do not respond due to high EPO level [93]. This has prompted the development of alternative therapeutic strategies (see Outstanding Questions). As with previous developments, these are underpinned by improved mechanistic understanding of MDS and its pathogenesis.

Localizing MDS erythroid differentiation blockage to the erythroblast stage steered development of luspatercept. Similarly, understanding the significance of splicing factors in MDS pathogenesis has driven the development of new therapeutics. Mutually exclusive heterozygous splicing factor point mutations are a frequent class of mutation in MDS patients. Each mutation has a distinct effect on splicing, but they could converge to produce adverse effects on hematopoiesis. Identifying and characterizing these mutant splicing factors enlightened the invention of various splicing modulators, such as pladienolides, FR901464, spliceostatin A, and mostly recently H3B-8800 which can selectively target spliceosomal mutant cells. Clinical evaluations of the specificity and efficacy of these modulators are ongoing and will be vital for LR-MDS treatment in the future.

Focusing on the causal relationship between BFU-E insufficiency and EPO resistance has led to the discovery of CHRM4 antagonists as potential agents to correct anemia and overcome EPO resistance in MDS. A number of clinical studies have reported that MDS patients exhibit significantly reduced levels of early erythroid progenitors or BFU-Es. As a consequence, erythropoiesis is compromised, and therapeutic benefit of conventional treatments is limited. A recent study has shown that CHRM4 is a regulator of BFU-E differentiation; inhibition of CHRM4 was found to drive BFU-E differentiation and increase the erythroid output in pre-clinical models. Efforts to gain a deeper understanding of CHRM4 regulation of erythropoiesis and clinical development of CHRM4 inhibitors are ongoing and potentially will open a new chapter in LR-MDS therapeutics.

Clinician’s Corner.

• A phase 1 multicenter clinical trial (NCT02841540^iv) of the splicing modulator H3B-8800 was completed recently and it was shown to be well-tolerated with escalated doses for prolonged time. H3B-8800 has also exhibited dose-dependent target engagement in MDS patients. 42% of MDS and other myeloid neoplasm transfusion dependent patients did not require RBC transfusion for ⩾8 weeks while participating in the study.

• Antisense oligonucleotide (ASO)-based treatments have been approved by the FDA recently for splicing related disorders such as spinal muscular atrophy and Duchenne muscular dystrophy. An ASO targeting STAT3 has been in phase 2 clinical trial (NCT02983578^v) for lung and colorectal cancers and also, it has shown efficacy in pre-clinical models of MDS [94].

• ESAs, such as erythropoietin and darbepoetin, are the first choice of drugs for LR-MDS patients. LR-MDS patients having endogenous EPO level <200 IU/L show beneficial responses to ESA treatment while patients with >500 IU/L EPO cannot receive ESAs and become RBC transfusion dependent.

• Enhancing the differentiation of BFU-Es with CHRM4 inhibitors provides a means of overcoming the limitation of ESAs by boosting erythropoiesis at an earlier stage in the process and independently of EPO. Pharmacological inhibition of CHRM4 has exhibited promising efficacy in pre-clinical models of MDS, correcting anemic phenotype, early erythroid progenitor insufficiency, abnormally elevated EPO level, and extending survival of splicing factor mutant MDS models. Overall, treatment with CHRM4 inhibitor was well tolerated and exhibited no side-effects on kidney and liver function [89]. Future development of CHRM4 inhibitors has the potential to target the vast majority of MDS patients.

Outstanding Questions.

• The splicing factor mutant cells rely heavily on remaining wild-type splicing activity. Can new and improved methods of splicing perturbation be developed?

• Are other GPCRs, besides CHRM4, functionally important in erythropoiesis?

• Can dysregulated GPCR signaling inform prognosis and efficacy of GPCR modulators?

• As a receptor for neurotransmitter acetylcholine (ACh), CHRM4 is involved in cholinergic signaling in the peripheral and central nervous system. What is the precise physiological and molecular mechanism of neural regulation of BFU-E differentiation?

• Various biological consequences converge to produce adverse effects on erythropoiesis in MDS. Ultimately will combinatorial regimens be effective in treating MDS?

Highlights.

• MDS is characterized by ineffective hematopoiesis arising from mutations in hematopoietic stem cells.

• Erythropoiesis stimulating agents are the first choice of drugs for low risk MDS patients but are often ineffective.

• Splicing factor mutations are the major class of mutation in MDS and recently developed splicing modulators can specifically target these mutations.

• Enhancing differentiation of early erythroid progenitors via inhibition of CHRM4, a newly identified negative regulator of BFU-E differentiation, has shown promising results in pre-clinical testing.

• Detailed investigation of nervous system regulation of HSPC differentiation could unravel novel regulatory mechanisms of hematopoiesis.

Glossary

Allogeneic hematopoietic stem cell transplantation (allo-HSCT)

a clinical procedure, used to treat a variety of blood cancers, in which healthy stem cells from a donor are transferred to a patient to replace their own stem cells.

International Prognostic Scoring System (IPSS)

a scoring system for staging MDS, which considers various risk factors, such as bone marrow blasts, chromosomal karyotype, number of cytopenias etc., for the prognosis of MDS.

Cytogenetics

a genetic study of the number and morphology of chromosomes inside the cell.

Erythropoietin (EPO)

a hormone produced by kidney that plays a crucial role in formation and maintenance of red blood cells.

International Unit (IU)

an internationally accepted unit of measurement for fat soluble substances such as hormones, vitamins, vaccines and drugs. of 8.4 micrograms of EPO is equivalent to 1000 IU.

Transfusion Independence (TI)

a state in which a patient’s body produces enough RBCs and does not rely on the exogenous supply of RBCs by transfusion.

Splicing modulators

small molecules that can selectively target and kill spliceosomal mutant cells, thereby inhibiting abnormal splicing.

Exonic splicing enhancer (ESE)

RNA sequences within exons that promote splicing of pre-mRNA to mature mRNA.

G protein-coupled receptor (GPCR)

the largest and most diverse group of transmembrane receptors which serve as the mediator between extracellular stimuli and intracellular signaling cascades.

Cholinergic receptor muscarinic 4 (CHRM4)

a GPCR of the central and peripheral nervous system that is activated by acetylcholine to bring about a variety of downstream functions.