Abstract
Immune evasion is a major obstacle for cancer treatment. Common mechanisms include impaired antigen presentation through mutations or loss of heterozygosity (LOH) of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1–3. However, in pancreatic ductal adenocarcinoma (PDAC), a malignancy refractory to most therapies including ICB4, mutations causing MHC-I loss are rarely found5 despite the frequent downregulation of MHC-I expression6–8. Here we find that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation through an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced MHC-I cell surface expression and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, autophagy inhibition restores surface MHC-I levels, leading to improved antigen presentation, enhanced anti-tumour T cell response and reduced tumour growth in syngeneic hosts. Accordingly, anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface MHC-I expression. Autophagy inhibition, either genetically or pharmacologically with Chloroquine (CQ), synergizes with dual ICB (anti-PD1 and anti-CTLA4), and leads to an enhanced anti-tumour immune response. Our findings uncover a role for enhanced autophagy/lysosome function in immune evasion through selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB as a therapeutic strategy against PDAC.
Results
MHC-I is enriched within autophagosomes and lysosomes
Human PDAC cell lines expressed heterogeneous levels of total MHC-I protein (Fig. 1a), and importantly, exhibited a punctate cytoplasmic distribution of MHC-I which co-localized with lysosomes (Fig. 1b). In contrast, non-transformed human pancreatic ductal epithelial (HPDE) cells showed predominant localization of MHC-I on the plasma membrane (Fig. 1b). Indeed, MHC-I molecules were highly enriched in PDAC lysosomes as compared to HPDE lysosomes (Fig. 1c, Extended Data Fig. 1a). Moreover, lysosomal inhibition resulted in MHC-I accumulation within lysosomes, confirming that MHC-I is actively routed to the lysosome for degradation (Fig. 1d). A substantial fraction of the MHC-I puncta also co-localized with LC3B-labeled autophagosomes in PDAC cells, consistent with the elevated autophagy levels in PDAC9–11 (Fig. 1e, Extended Data Fig. 1b). Notably, similar phenotypes were observed in several non-small-cell lung cancer (NSCLC) cell lines (Extended Data Fig. 1c,d). Flow cytometry-based analysis of total intracellular versus plasma membrane MHC-I confirmed a higher relative abundance of intracellular MHC-I in the majority of PDAC cell lines (Fig. 1f). Similarly, surface MHC-I levels were lower in PDAC cells derived from a genetically engineered mouse model (GEMM) of PDAC12 than normal pancreas cells (Extended Data Fig. 1e). Furthermore, immunofluorescence staining revealed that all human PDAC tumours analyzed contained significant regions with intracellular MHC-I localization (Fig. 1g, Extended Data Fig. 1f), supporting our in vitro findings. Together, these data suggest that MHC-I molecules are reduced at the cell surface and predominantly localized within autophagosomes and lysosomes in PDAC. Indeed, autophagy inhibition by ATG3 and ATG7 knockdown as well as lysosomal inhibition with Bafilomycin A1 (BafA1), increased total and plasma membrane MHC-I levels in PDAC cells (Fig. 2a,b, Extended Data Fig. 2a–i). Moreover, surface MHC-I levels of Atg5−/− mouse PDAC cells10 were higher than those of Atg5+/+ PDAC cells (Extended Data Fig. 2j). Importantly, lysosomal inhibition with BafA1 or chloroquine (CQ) increased MHC-I proteins but did not affect those involved in antigen processing and presentation (Extended Data Fig. 2k,l), suggesting that autophagy inhibition would not impair these steps. Similar phenotypes were also observed in several NSCLC cell lines (Extended Data Fig. 2m–o).
Recent studies have revealed non-canonical functions for a subset of autophagy proteins in alternate trafficking pathways such as LC3-associated phagocytosis (LAP)13 and endocytosis (LANDO)14. Knockdown of FIP200, which is required for autophagy but not for LAP/LANDO, led to an increase in MHC-I levels (Extended Data Fig. 3a–c). Similarly, knockdown of ATG14, Atg13 or Ulk1, all required for autophagy but dispensable for LAP, increased surface MHC-I levels (Extended Data Fig. 3d–j). In contrast, no obvious increase in surface MHC-I was observed upon knockdown of Rubicon, which is required for LAP/LANDO but not for autophagy (Extended Data Fig. 3k,l). Collectively, these data suggest a specific role for macroautophagy in the trafficking of MHC-I to the lysosome.
NBR1 mediates selective autophagy of MHC-I
Recent studies have revealed that autophagy can selectively degrade target molecules using autophagy cargo receptor proteins that bind to and recruit substrates to autophagosomal membranes15. To identify autophagy receptor protein(s) involved in MHC-I degradation in PDAC cells, we generated a proximity biotinylation assay whereby HLA-A was fused to the biotin ligase, TurboID (TrID)16 and Flag (HLA-A-TrID). Upon biotin addition, HLA-A-TrID covalently tags endogenous proteins within a few nanometers of the ligase with biotin (Extended Data Fig. 4a,b). Among the autophagy receptors tested, only NBR1 showed significant biotinylation, indicating that NBR1 interacts with MHC-I (Fig. 2c). Additionally, immunofluorescence revealed more frequent co-localization between NBR1 and MHC-I in PDAC cells relative to HPDE cells (Fig. 2d). NBR1 has been shown to interact with and target ubiquitylated substrates for degradation17. Indeed, MHC-I is poly-ubiquitylated in PDA cells (Fig. 2e, Extended Data Fig. 4c) while LC3B is not ubiquitylated and EGFR is mono-ubiquitylated as previously described18. Accordingly, NBR1 lacking its ubiquitin-associated (UBA) domain failed to co-localize with MHC-I, despite retaining localization with LC3B (Fig. 2f, Extended Data Fig. 4d). Finally, NBR1 knockdown increased total and plasma membrane MHC-I levels in PDAC cells (Fig. 2g,h, Extended Data Fig. 4e,f), confirming a role for NBR1 in MHC-I regulation. Together, these data demonstrate that surface MHC-I is decreased in PDAC via an NBR1 mediated autophagy-lysosomal pathway.
Autophagy inhibition enhances anti-tumour immunity
CD8+ cytotoxic T cells play critical roles in anti-tumour immunity. We hypothesized that reduced surface MHC-I levels on PDAC cells may facilitate their evasion from CD8+ T cells, which recognize tumour antigens presented by MHC-I. To test this, we used mouse PDAC cells derived from C57Bl/6 mice and engineered them to express a doxycycline (Dox)-inducible dominant-negative mutant of Atg4B (Atg4BC74A), which potently inhibits autophagy12,19. Dox treatment efficiently inhibited autophagy and increased surface MHC-I levels (Extended Data Fig. 5a,b) but did not affect other cell surface molecules, such as those that are immune inhibitory (Extended Data Fig. 5c). These data support a specific mechanism that links surface MHC-I levels and autophagy.
To evaluate antigen presentation, the model neoantigen ovalbumin (OVA) was expressed in mouse PDAC cells carrying Dox-inducible Atg4BC74A (Extended Data Fig. 5d). Dox treatment increased surface expression of both MHC-I and OVA-derived peptide SIINFEKL bound to H-2Kb, confirming enhanced peptide presentation (Fig. 3a,b, Extended Data Fig. 5e,f). OVA-specific CD8+ T cells (OT-I cells) co-cultured with Dox-pretreated PDAC cells showed higher proliferation and expression of Interferon-γ and TNF-α than those co-cultured with non-pretreated PDAC cells (Fig. 3c, Extended Data Fig. 5g,h). Accordingly, Dox-pretreated PDAC cells showed reduced viability upon co-culture with OT-I cells (Fig. 3d), indicating enhanced T cell mediated tumour cell killing. Importantly, these effects were MHC-I specific, as a SIINFEKL-H-2Kb blocking antibody partially inhibited OT-I proliferation and rescued PDAC cell viability in the setting of autophagy inhibition (Fig. 3c,d). Collectively, these results indicate that tumour-specific autophagy inhibition leads to increased antigen presentation, which enhances CD8+ T cell proliferation, activation and tumour cell killing in vitro.
To test the impact of autophagy inhibition on anti-tumour immune responses in vivo, mouse PDAC cells expressing Dox-inducible mStrawberry (mSt) or mSt-Atg4BC74A (4B) (Extended Data Fig. 6a) were orthotopically transplanted into syngeneic (C57BL/6) mice. Autophagy-inhibited cells (4B) formed smaller tumours with higher MHC-I expression than control cells (mSt) while PD-L1 expression was unchanged (Fig. 3e–g, Extended Data Fig. 6b–f and 7k). Moreover, autophagy-inhibited tumours (4B) exhibited a significant increase in infiltrating CD8+ T cells and a decrease in myeloid derived suppressor cells (MDSC), a major immunosuppressive cell type in PDAC tumours (Fig. 3h, Extended Data Fig. 6g–j)20. Similarly, knockdown of Atg7 resulted in a significant reduction in tumour burden and an increase in tumour-infiltrating T cells (Extended Data Fig. 6k–n). Notably, there was a significant correlation between smaller tumour sizes and increased CD8+ T cell infiltration (Extended Data Fig. 6o), supporting a role of T cell immunity in control of autophagy-deficient tumours. Similar results were obtained in a liver metastasis model: mice injected with autophagy-inhibited cells (4B) exhibited lower metastatic burden, higher MHC-I expression on cancer cells, and more tumour-infiltrating CD8+ T cells than mice injected with control cells (mSt) (Fig. 3i–l, Extended Data Fig. 6p–t). Together, these results confirm that autophagy-inhibition restores surface MHC-I levels on cancer cells and enhances anti-tumour T cell response in vivo.
Importantly, antibody-mediated CD8+ T cell depletion restored the growth of autophagy-inhibited tumours (4B) (Fig. 3m, Extended Data Fig. 7a–c), confirming the role of CD8+ T cells in tumour control. We also find that mice deficient for CD103+ dendritic cells (DCs; Batf3−/−)21, which play pivotal roles in CD8+ T-cell priming22 and recruitment into tumours20,23, showed almost complete loss of tumour-infiltrating CD8+ T cells (Fig. 3n, Extended Data Fig. 7d–f). Accordingly, growth of autophagy-inhibited tumours (4B) was restored in Batf3−/− mice (Fig. 3o). These results indicate that the anti-tumour effects of tumour-specific autophagy inhibition are mediated, at least in part, by CD8+ T cells, and this process requires CD103+ DCs.
Finally, to confirm the impact of increased MHC-I expression following autophagy inhibition on tumour growth in vivo, cell surface MHC-I was depleted by knocking down beta-2 microglobulin (B2m), a critical component of the MHC-I complex (Extended Data Fig. 7g). B2m knockdown led to MHC-I depletion in vivo, decreased the number of CD8+ T cells in autophagy-inhibited tumours (4B), and rescued the growth of autophagy-inhibited tumours (4B)(Fig. 3p–r, Extended Data Fig. 7h–k). We also found that tumour-infiltrating CD103+ DCs, which are increased in autophagy-inhibited tumours (4B), were decreased following B2m knockdown (Extended Data Fig. 7f,j). This is in line with a recent study showing that MHC-I-restricted antigen-recognition by CD8+ T cells can trigger DC activation, which further augments CD8+ T cell recruitment into the tumour24. Overall, these data indicate that increased surface MHC-I expression on PDAC cells following tumor-specific autophagy inhibition is a prerequisite for increased CD8+ T cell infiltration and tumor cell killing.
Basal autophagy determines immunogenicity
Our data prompted us to hypothesize that basal autophagy flux might determine the immunogenicity of PDAC cells. Mouse PDAC cells grown as organoids25 exhibited considerable heterogeneity in autophagy flux, which closely resembled that of orthotopic tumours (Extended Data Fig. 8a, b). Taking advantage of this heterogeneity, PDAC cells with the lowest and highest 20% of GFP/RFP ratio were isolated as the autophagy-high (AThi) and -low (ATlo) cells (Extended Data Fig. 8c). Transcriptome analysis confirmed upregulation of autophagy/lysosome related genes and the MiT/TFE transcription factors, master regulators of autophagy/lysosome gene expression11, in the AThi population (Extended Data Fig. 8d,e). Importantly, AThi and ATlo cells derived from a clonal population showed a similar transcriptional profile (Extended Data Fig. 8f), suggesting that diversities in basal autophagy flux arise not from genetic variations but instead from heterogeneous expression of the autophagy/lysosome gene program11.
Consistent with the cell-autonomous roles of autophagy in PDAC9,10, ATlo cells exhibited reduced clonogenic capacity in vitro (Extended Data Fig. 8g,h), a result similar to the decreased clonogenic growth of Atg4BC74A expressing cells (Extended Data Fig. 8i). Upon orthotopic transplantation into syngeneic hosts, ATlo cells gave rise to smaller tumours than AThi cells (Extended Data Fig. 9a). Importantly, this was reproduced using cells sorted from a clonal population (Extended Data Fig. 9b), suggesting that tumour-cell intrinsic factors account for the observed phenotypes rather than differences in retroviral-vector integration sites or copy numbers of the reporter. Furthermore, ATlo-derived tumours exhibited higher MHC-I expression and more tumour-infiltrating CD8+ T cells, which inversely correlated with tumour weight, compared to AThi-derived tumours (Extended Data Fig. 9c–f). This growth advantage of AThi-derived tumours was lost in nude mice (Extended Data Fig. 9g), confirming a role of T cells in the suppression of ATlo-derived tumours. In the liver metastasis model, mice injected with ATlo cells exhibited lower metastatic burden and higher MHC-I expression on PDAC cells than mice injected with AThi cells (Extended Data Fig. 9h–j). Notably, CD8+ T cells tended to accumulate around PDAC cells with low-autophagy flux, while CD8+ T cells were scarce around PDAC cells with high-autophagy flux (Extended Data Fig. 9k). These results, along with the data in autophagy inhibition models, indicate that autophagy is a critical determinant of immunogenicity in PDAC cells.
Autophagy inhibition sensitizes PDAC to dual ICB
PDAC is refractory to immune checkpoint blockade (ICB)4. To test if autophagy inhibition might sensitize PDAC to ICB, we treated established syngeneic orthotopic tumours with anti-PD-1 antibody alone or dual ICB (anti-PD-1/CTLA4 antibodies). Consistent with a recent clinical trial4, control tumours (mSt) did not respond to either treatment. In contrast, autophagy-inhibited tumours (4B) responded significantly to dual ICB (Fig. 4a–c), but not to anti-PD-1 antibody alone (Extended Data Fig. 10a–c). These tumours showed increased infiltration with bulk CD8+ T cells and PD1+Tim3− cells (Fig. 4d, Extended Data Fig. 10d–g), which retain greater function than the most severely exhausted/dysfunctional PD1+Tim3+ cells26. These results indicate that tumour-specific autophagy inhibition sensitizes PDAC tumours to dual ICB.
Finally, we assessed the translatability of our findings to systemic autophagy inhibition using chloroquine (CQ), a clinically available antimalarial agent that inhibits acidification of the lysosome and has been used to inhibit autophagy in patients27–29. Treatment with lysosomal inhibitors CQ or BafA1 increased surface MHC-I levels in mouse PDAC cells in vitro (Extended Data Fig. 10h). CQ treatment also increased surface expression of MHC-I, but not PD-L1, in orthotopic tumours (Extended Data Fig. 10i–k). However, CQ monotherapy failed to significantly reduce tumour weight or increase T cell infiltration (Extended Data Fig. 10l,m), possibly due to the unfavorable pharmacokinetics of this drug in vivo27. Strikingly, the combination of CQ and dual ICB (CQ + ICB) exerted potent anti-tumour activity (Fig. 4e–i) and a reduction in autophagy flux was confirmed in CQ-treated tumours (Fig. 4j, Extended Data Fig. 10n). Moreover, CQ + ICB treated tumours exhibited increased CD8+ T cell infiltration (Fig. 4k) and an increase in functional PD-1+Tim3− CD8+ T cells (Extended Data Fig. 10o). Overall, these data indicate that autophagy inhibition, either in cancer cells alone or systemically, sensitizes PDAC tumours to dual ICB (Fig. 4l).
Discussion
Our results suggest that autophagy is a critical regulator of immunogenicity in PDAC cells. This is in line with a recent study showing the lysosomal pathway as strongly correlated with reduced CD8+ T cell infiltration in human PDAC30. Additionally, autophagy-related genes are enriched in MHC-I negative PDAC cells that reside in liver metastasis8, also suggesting a role for autophagy as a negative regulator of MHC-I. Given the critical roles for the autophagy/lysosome system in supporting PDAC metabolism and growth9,11, our data on immune evasion adds to the growing list of cell-autonomous and non-cell-autonomous functions of the autophagy/lysosome system in PDAC pathogenesis10,12.
We found that systemic autophagy inhibition by CQ, as well as tumour-specific autophagy inhibition, sensitizes PDAC to dual ICB. In addition, recent evidence accounts for the improved therapeutic effects of systemic autophagy inhibition: First, host autophagy supports tumour growth by providing nutrients31–33. Second, loss of autophagy proteins or CQ treatment increases surface MHC-I levels in dendritic cells, leading to enhanced CD8+ T cell response in virus infection models34. Also, autophagy inhibition directly enhances anti-tumour activity of CD8+ T cells35. Lastly, loss of LAP, a process also inhibited by CQ, polarizes tumour-associated macrophages (TAMs) into a tumour suppressive phenotype (M1), promoting anti-tumour T cell responses13. In line with these, recent clinical trials have shown that hydroxychloroquine, a derivative of CQ, has activity in PDAC patients28,29. Whether the addition of ICB would be synergistic remains to be determined.
In this study, we focused on CD8+ T cells, given their direct interaction with MHC-I on cancer cells. However, we also observed changes in other immune cells such as MDSCs, CD4+ T cells and CD103+ DCs upon autophagy inhibition. Investigating how these and changes in other immune cell types are mediated, such as the potential involvement of secreted proteins from PDAC cells upon autophagy inhibition, will be important subjects of future work.
Despite the evidence mentioned above and recent work demonstrating that autophagy inhibition does not impair anti-tumour adaptive immunity36, autophagy inhibition has also been reported to effect some aspects of the immune system such as memory formation in virus specific CD8+ T cells and CD4+ T cell priming by DCs37,38 and may impair chemotherapy-induced immunogenic cell death39. Therefore, further studies will be needed to define more subtle aspects of the immune response upon autophagy inhibition and how to best combine autophagy/lysosome blockade with cytotoxic and immune based therapies. For instance, deeper insights into the potential impact of basal immunogenicity40 on response to autophagy inhibition, the differences in mutational burden in mouse and human tumors41, the impact of potential dominant antigens in experimental systems41, and the heterogeneity described here in terms of MHC-I expression in patients will likely contribute to the successful clinical translation of our findings. Importantly, the mechanistic insights described here regarding how autophagy can promote immune evasion, provide strong rationale to pursue these studies with the ultimate goal of developing new therapeutic approaches for PDAC patients.
Methods
Cell culture
The cell lines PaTu-8988T, KP4, MiaPaca2, Panc 2.03, PaTu-8902, Panc1, AsPc1, HupT3, A549 were obtained from the American Type Culture Collection (ATCC) or the DSMZ. H441, H358, HCT116 and BEAS-2B were provided by Eric Collisson, UCSF. HPDE was provided by Ming Tsao42. Cells were cultured in the following media: PaTu-8988T, KP4, MiaPaca2, PaTu-8902, Panc1, AsPc1 and BEAS-2B in DMEM supplemented with 10% FBS; Panc 2.03 and HupT3 in RPMI with 10% FBS; HPDE cells were cultured in keratinocyte serum-free (KSF) medium supplemented by epidermal growth factor and bovine pituitary extract (Life Technologies, Inc., Grand Island, NY), supplemented with 1% Pen/Strep (Gibco). Cell lines were regularly tested and verified to be mycoplasma negative using MycoAlert Detection Kit (Lonza) or via PCR.
Primary murine PDAC cell lines were established from pancreatic tumours in respective GEMMs as described previously12. HY15549 and HY19636 cells were established from female KPC mice (p48-Cre+, KrasLSL-G12D/+, Trp53lox/+)43 that were fully backcrossed into a C57BL/6 background. The other cell lines were derived from the following animals10,12: Atg5+/+ KPC cells, Pdx1-Cre+, KrasLSL-G12D/+, Trp53lox/+, Atg5+/+ mice; Atg5−/− KPC cells, Pdx1-Cre+, KrasLSL-G12D/+, Trp53lox/+, Atg5lox/lox mice; AY6284 cells, p48-Cre+, KrasLSL-G12D/+, Trp53lox/+, RosaLSL-rtTA, mSt-Atg4BC74A mice12. All mice PDAC cells were maintained in DMEM (Corning) supplemented with 10% FBS (Atlanta Biologicals S11550H) and 1% Pen/Strep (Gibco).
Cells were grown in a humidified incubator with 5% CO2 at 37°C. Cultures were routinely verified to be negative for mycoplasma. Cell lines were authenticated by fingerprinting, and low passage cultures were carefully maintained in a central lab cell bank.
Constructs
GFP-NBR1 and GFP-NBR1 dUBA were provided by Jayanta Debnath. LysoTag - TMEM192-mRFP-3xHA (TMRHA) - was generated by subcloning the cDNA of TMEM192 (Origene) together with monomeric Red Fluorescent Protein (mRFP) and 3xHA tag into the NheI and EcoRI sites of pLJM1 lentiviral vector. HLA-A-TurboID-FLAG (HLA-A-TrID) was generated by subcloning the cDNA of HLA-A (Addgene plasmid, #85162) into the EcoRI and NotI sites of the TurboID pLVX vector (gift from Roberto Zoncu, UC Berkeley). pMXs GFP-LC3-RFP was a gift from Noboru Mizushima (Addgene, plasmid #117413). For Dox-inducible expression of Atg4BC74A, mTurquoise2 or mStrawberry was fused to Atg4BC74A and inserted into either pSLIK-Hygro (used for in vitro studies) (Addgene, plasmid #25737) or pINDUCER20 (used for in vivo studies) (Addgene, plasmid #44012), using the Gateway Cloning system (Thermo Fisher Science). For the generation of OVA-expressing cells, the cOVA fragment was cloned from pCI-neo-cOVA (Addgene, plasmid #25097), fused with 2A peptide and mStrawberry sequences using NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs) according to manufacturer’s instruction, and inserted into the EcoRI and SalI sites of pBabe-zeo (Addgene, plasmid #1766) to generate pBabe-cOVA-2A-mStrawberry. Stable cOVA expression was confirmed and monitored with mStrawberry fluorescence.
shRNAs
shRNA vectors (pLKO.1 puro) were obtained from the Sigma MISSION TRC shRNA library. The sequences and RNAi Consortium clone IDs for the shRNAs used are as follows:
shATG3#1 (human): 5’-GATGTGACCATTGACCATATT-3’ (TRCN0000148120); shATG3#2 (human): 5’-GCTGTCATTCCAACAATAGAA-3’ (TRCN0000147381); shATG7#1 (human): 5’-CCCAGCTATTGGAACACTGTA-3’ (TRCN0000007587); shATG7#2 (human): 5’-GCCTGCTGAGGAGCTCTCCAT-3’ (TRCN0000007584); shNRB1#1 (human): 5’-GCTTCATAGTTATTTGGCATT-3’ (TRCN0000123159); shNBR1#2 (human): 5’-GCAGCATTTGTGGATGAGAAT-3’ (TRCN0000123160); shNBR1#3 (human): 5’GCCAGGAACCAAGTTTATCAA-3’ (TRCN0000123161); shFIP200#1 (human): 5’- GCACTCTTTAACACATTCTTT-3’ (TRCN0000013523); shFIP200#2 (human): 5’- GCTGTGAATGAGTTTGTAATA-3’ (TRCN0000013524); shATG14#1 (human): 5’-CCATAGAACTTGGTCATGTTT-3’ (TRCN0000144080); shATG14#2 (human): 5’-GATCAATTACAACCACTGCAT-3’ (TRCN0000145367); shAtg3#1 (mouse): 5’-CATATCACAACACAGGTATTA-3’ (TRCN0000247440); shAtg3#2 (mouse): 5’- GTACATCACTTACGACAAATA-3’ (TRCN0000247442); shAtg7#1 (mouse): 5’-TTCTGTCACGGTTCGATAATG-3’ (TRCN0000305991); shAtg7#2 (mouse): 5’- GCCAACATCCCTGGATACAAG-3’ (TRCN0000375444); shAtg7#3 (mouse): 5’- TCTTACCCTGCTCCATCAAGA-3’ (TRCN0000375421); shB2m (mouse): 5’-CCAGTTTCTAATATGCTATAC-3’ (TRCN0000295705); shAtg13#1 (mouse): 5’-TGAAGTCTCTTCTCGCTATTA-3’ (TRCN0000277121); shAtg13#2 (mouse): 5’-GACATACCTTTCGCCATGTTT-3’ (TRCN0000176029); shUlk1#1 (mouse): 5’-CGCTTCTTTCTGGACAAACAA-3’ (TRCN0000319764), shUlk1#2 (mouse): 5’-CGCTTCTTTCTGGACAAACAA-3’ (TRCN0000028768); shGFP: 5’-TGCCCGACAACCACTACCTGA-3’ (TRCN0000072186). shGFP and shScr (Addgene, plasmid #17920) were used as controls.
siRNAs
The siRNAs used in this study are: siNC (Silencer Negative Control #1 siRNA, Thermo, Cat #13778030), siRubicon #1 (s104762, Ambion), and siRubicon #2 (s104763, Ambion). Cells were transfected with siRNAs using Lipofectamine RNAi Max Transfection Reagent (Life Technologies).
Retroviral and lentiviral transduction
For the transfection of pMXs GFP-LC3-RFP44 and pBabe-cOVA-2A-mStrawberry, retrovirus was produced by co-transfection of HEK293FT cells with a retroviral vector and the packaging plasmids pHit60 and VSVG at a 0.5:0.25:0.25 ratio. For the transfection of lentiviral vectors (pSLIK-hygro, pINDUCER20, and pLKO.1-puro), lentivirus was produced by co-transfection of HEK293FT cells with a lentiviral vector and the packaging plasmids psPAX2 (Addgene, plasmid #12260) and pMD2.G (Addgene, plasmid #12259) at a 0.5:0.25:0.25 ratio. Transfection was performed using Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to manufacturer’s instructions. The viral supernatant was collected 48 hrs after transfection, filtered through a 0.45 μm filter, and used for infection together with Polybrene reagent (EMD Millipore). Cells expressing Dox-inducible constructs were sorted for positive fluorescent expression upon Dox treatment to select inducible cells, and then sorted for no fluorescent expression after Dox withdrawal to remove cells with leaking expression, as described previously8.
Immunofluorescence
Human cell lines were cultured for two days on coverslips coated with fibronectin. After two PBS washes, cells were fixed and permeabilized with paraformaldehyde for 15 min at room temperature or ice-cold methanol for 5 min at −20°C. PFA fixed cells were permeabilized with 0.1% Saponin. Samples were then blocked with 5% normal goat serum for 15 min at room temperature prior to incubation with primary antibodies (Supplementary Table 1) overnight at 4°C. After washing three times with PBS, cells were incubated in secondary antibody at room temperature for 20 min. Slides were mounted on glass slides using DAPI Fluoromount-G (0100–20, SouthernBiotech) and imaged on a Zeiss Laser Scanning Microsope (LSM) 710 using a 63x objective. Image processing and quantification were performed using ImageJ.
Lysosome immunoprecipitation
For lysosome immunoprecipitation experiments, HPDE and human PDAC cell lines stably expressing TMRHA were ruptured and intact lysosomes from 1–2 mg of total protein per sample was immunoprecipitated using HA-conjugated Dynabeads as previously described45,46.
Poly-Ubiquilin UBA affinity capture
Cells were washed twice with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % NP-40, 10 mM N-ethylmaleimide) supplemented fresh with protease inhibitor cocktail. Lysates were incubated at 4˚ C for 15 min and clarified by centrifugation at 20,000 xg at 4˚ C for 15 min. Samples were quantified by BCA Protein Assay Kit and diluted to 1 mg/mL with Dilution Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, protease inhibitor). 1–1.5 mg of protein lysates were incubated with 50 μl of Ubiquilin 1 Tandem UBA Agarose (BostonBiochem AM-130) overnight at 4˚ C. Samples were then washed 3x in High Salt Wash Buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.5 % NP-40) and once with 10 mM Tris-HCl, pH 7.5. Samples were eluted by adding Laemlli buffer and incubating at 65˚ C for 15–20 min.
For DUB digestions, affinity captured material was washed 1x with DUB digestion buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 20mM DTT). Liquid was removed and beads were resuspended in 20ml of DUB digestion buffer and 5mg of USP2 catalytic domain (Usp2cc; BostonBiochem E-506) for 1hr with gentle shaking at 30˚ C. Beads were washed once in High Salt Wash Buffer and once with 10 mM Tris-HCl, pH 7.5 prior to eluting affinity captured material by adding Laemmli buffer and incubating at 65 ˚ C for 15min.
Proximity biotinylation
Human PDAC cells stably expressing HLA-A-TrID were cultured in DMEM supplemented with 10% dialyzed FBS (DMEM + dFBS) for 48 h. Cells were incubated with 10 μM Biotin (Sigma) and incubated at 37˚ C for 30 min. Media was replaced with DMEM + dFBS and incubated at 37˚C for a further 2–3 hrs. For negative controls, we omitted exogenous biotin. Cells were then washed 2x in ice-cold PBS and lysed in ice-cold lysis buffer (1 % Triton X-100, 130 mM NaCl, 2.5 mM MgCl2, 2 mM EGTA, 25 mM HEPES pH 7.4, supplemented fresh with protease inhibitor cocktail) for 30 min on ice. Samples were clarified by centrifugation at 13,300 rpm for 10 min at 4˚ C. Protein content was measured using BCA Protein Assay Kit (Life Technologies 23227). 1–2 mg of protein lysates were incubated with 50 μl of Dynabeads MyOne Streptavidin C1 (Life Technologies) overnight. The beads were washed twice in Wash Buffer 1 (2 % SDS in dH2O), once in Wash Buffer 2 (0.1 % deoxycholate, 1 % Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM HEPES, pH 7.5), once in Wash Buffer 3 (250 mM LiCl, 0.5 % NP-40, 0.5 % deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1), and twice in Wash Buffer 4 (50 mM Tris, pH 7.4, and 50 mM NaCl). Washes were performed at RT for 5 min with gentle agitation. Samples were eluted in Laemlli buffer and boiled at 95˚ C. Quantification of biotinylation activity was measured as intensity of each lane in the +/−Biotin immune-precipitation condition divided by the corresponding intensity of the ligase expression band in the input. +Biotin ratios were then normalized to the -Biotin control ratio and to background.
Organoid culture
Murine normal pancreatic organoids were established as described previously25,47 with slight modifications. Briefly, pancreas was harvested from female C57BL/6 mice (4–10 weeks of age), mechanically minced with scissors, and digested with 1 mg/mL Collagenase P (Sigma), 4 mg/mL Dispase II (Sigma), 10 mM HEPES (Thermo Fisher Scientific), 1% FBS, 1 mg/mL Trypsin Inhibitor from Soybean (Sigma) in DMEM for 20 min at 37°C. Digested tissues were embedded in Matrigel (Corning 356231) and cultured in Advanced DMEM (Thermo Fisher Scientific) supplemented with 200 mM L-Glutamine (Thermo Fisher Scientific), 10 mM HEPES (ThermoFisher Scientific), 1% Antibiotic-Antimycotic (Thermo Fisher Scientific), 500 ng/mL recombinant murine Rspondin-1 (Peprotech), 50 ng/mL recombinant murine EGF (Peprotech), 100 ng/mL recombinant murine Noggin (Peprotech), 1 μM Jagged-1 (188–204) (AnaSpec, AS-61298), 100 μg/mL Trypsin inhibitor from soybean (Sigma), and 10 μM Y-27632 (Enzo). Two days before analysis, culture media was replaced with DMEM supplemented with 10% FBS. PDAC organoids were generated by embedding murine PDAC cell lines in Matrigel and were grown in DMEM with 10% FBS. For the dissociation of organoids for further assays, organoids were digested with TrypLE (ThermoFisher, A1217701) solution at a 2x concentration for 20 min at 37°C, followed by filtration through a 40-μm nylon strainer.
Quantitative proteomics
Quantitative mass spectrometry-based proteomics was performed as previously described48,49 based on the SL-TMT workflow50. Briefly, cells were lysed in a lysis buffer (200mM HEPES pH 8.5, 8M Urea, 1x Complete Protease Inhibitor Cocktail (Roche), 1× PhosStop (Roche)) and homogenized by passing through a 21-gauge needle. Lysates were collected by centrifuging at 20,000 × g for 5 min at 4 °C, followed by disulfide bond reduction with 5 mM dithiothreitol at 37°C for 25 min and alkylation with 10mM iodoacetamide at room temperature for 30 min in the dark. Chloroform–methanol precipitation of protein was performed, followed by protease digestion in HEPES buffer (200mM, pH 8.5). Each sample containing 100 μg protein was digested at a 1:100 protease-to-protein ratio with LysC protease at room temperature overnight, followed by digestion with trypsin at 37°C for 6 hrs. Approximately 50 μg of peptides from each sample was labelled with 100 μg TMT reagent which were dissolved in anhydrous ACN to achieve a final concentration of 30 % (v/v). TMT labelled samples was acidified, vacuum centrifuged to near dryness and subjected to C18 SPE (Sep-Pak, Waters). Samples were subjected to basic pH reversed-phase HPLC. Data were obtained with Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) coupled with a Proxeon EASY-nLC 1000 LC pump (Thermo Fisher Scientific). Peptide separation was done using a custom Accucore C18 resin (2.6 μm, 100Å, ThermoFisher Scientific) column for 3 hrs using a gradient of 6–30% ACN in 0.125% formic acid with a flow rate of 300 nl/min. All analysis involved an MS3-based TMT method as previously mentioned51 and mass spectra was processed as described earlier48.
Tumour cell and OT-I cell co-culture experiment
Mouse PDAC cells stably expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4BC74A were grown as organoids, treated with or without Dox (1 μg/mL) for 96 hrs. Organoids were dissociated into single cells, which were then incubated with either anti-H-2Kb-SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C. Total splenocytes were harvested from OT-I mice and CD8+ T cells were enriched using Dynabeads® Untouched™ Mouse CD8 Cells (Invitrogen, 11417D) following manufacturer’s instructions. Isolated CD8+ T cells were labelled with 10 μM CFSE (BioLegend) for 10 min at RT in the dark, washed 3x with RPMI-1640 supplemented with 10% FBS. Ten thousand PDAC cells and forty thousand CD8+ cells were seeded in 96-well plates and cultured in 100 μL 50% DMEM and 50% RPMI-1640 supplemented with 10% FBS, 10 ng/mL recombinant murine IL-2 (Peprotech), 27.5 μM 2-Mercaptoehanol (Gibco), and 100 μg/mL of respective antibodies. After 48 hrs, CD8+ T cells were harvested and stained with anti-CD8a antibody (AF647, clone 53–6.7, BioLegend) and DAPI, and proliferation was analyzed by CFSE dilution using flow cytometry. After removal of CD8+ T cells, the viability of remaining PDAC cells were measured by CellTiter-Glo (Promega).
Clonogenic assay
For 2D clonogenic assays9, cells were plated in 6-well plates at 300 cells per well in 2 mL DMEM supplemented with 10% FBS. After 7 days, colonies were fixed with 80% methanol, stained with 0.2% crystal violet, and counted. For 3D clonogenic assay, single cells were sorted directly into 384-well round bottom Ultra-Low Attachment plates (Corning 3830) and grown in DMEM supplemented with 10% FBS and 2% Matrigel (Corning 356231). After 10 days, the number of wells with spheroids were counted.
Mice
Female 8–10 week old C57BL/6 mice or NCr nude (B6NTac, Taconic) were used for allograft experiments. OT-I transgenic mice (Cat# 003831) and Batf3−/− mice (Cat# 013755) were purchased from Jackson Laboratory. All mice were bred and maintained in the animal facility of the New York University School of Medicine. All animal procedures were approved by the New York University School of Medicine Institutional Animal Care and Use Committee (IACUC) under protocol numbers IA16–00507 and IA16–01331.
Animal experiments
Orthotopic and intrasplenic injections of PDAC cells were performed as described previously31,52. Briefly, mice were anesthetized by an intra-peritoneal (i.p.) injection of ketamine and xylazine. A small incision was made on the upper left quadrant of the abdomen, and either the pancreas or the spleen was externalized. For orthotopic injection, cells were suspended in 20 μL of Matrigel (Corning 356231): HBSS (1:1) solution and injected into the pancreatic tail with insulin syringes (29-gauge needle, BD 324702). 1 × 104 HY15549 cells or 2 × 104 HY19636 cells were injected unless otherwise indicated. For intrasplenic injection, 1 × 106 cells were suspended in 100 μL HBSS and then drawn into an insulin syringe (28-gauge needle, BD 329461) which were pre-loaded with 200 μL HBSS. The externalized spleen was divided by ligating clips (Teleflex, #002200), and cells were injected into the hemispleen. After injection, splenic vein was ligated with ligating clips (Teleflex, #001200) at the hilum of the spleen, and then the hemispleen was removed. After the procedures, the peritoneum was closed with a 3–0 VICRYL VIOLET suture (Ethicon, J311H), and the skin was closed using the BD AutoClip Wound Closing System (BD). For some experiments, mice were fed with Dox-containing diet (625mg/kg) for the indicated period. Mice were euthanized at the indicated time points and tumours or the liver were collected after trans-cardiac perfusion with PBS.
For CD8+ T cell depletion, mice received i.p. injection of anti-mouse CD8a antibody (200 μg, clone 53–6.7, Cat# BE0004–1) or isotype control (200 μg, clone 2A3, Cat# BE0089). For immune checkpoint blockade experiments, mice received i.p. injection of anti-mouse PD-1 antibody (200 μg, clone RMP1–14, Cat# BE0146) and anti-mouse CTLA-4 antibody (200 μg, Cat# BE0131), or rat IgG2a isotype control (200 μg, clone 2A3, Cat# BE0089) and polyclonal Syrian hamster IgG (200 μg, Cat# BE0087). All antibodies used in in vivo experiments were obtained from BioXCell. For CQ treatment, mice received i.p. injection of CQ solution in PBS at 60 mg/kg or PBS every day starting day 4 or 5 after tumour cell implantation. Mice were randomly assigned to specific treatment groups at the beginning of treatment.
All experiments were carried out in a clean conventional facility, where mice were housed in pre-packaged disposable irradiated cages, and fed with irradiated diet and acidified water. Microisolator cages were located on ventilated racks. No tumours in the mice exceeded IACUC-defined maximum diameters of > 2 cm. Sample sizes were determined based on our preliminary experiments and no sample size calculation was done. Blinding was not perfomred as the investigator needed to know the treatment groups in order to perform study. Tumour weights (an objective measurement) were measured only at the study endpoints after mice were euthanized and tumours were harvested.
Flow cytometry
For surface and intracellular MHC-I staining of human cell lines, cells were stained with Alexa Fluor® 488 anti-human HLA-A, B, C antibody (BioLegend, clone W6/32) at a 1:75 dilution for 45 minutes at 4°C in the dark and washed with PBS plus 2% FBS and 2mM EDTA (FACS buffer). Cells were then fixed and permeabilized prior to staining with PE conjugated anti-human HLA-A, B, C antibody (BioLegend, clone W6/32) at a 1:75 dilution for 45 min at room temperature and washed with FACS buffer. For cell surface molecule staining of mouse cells, single cell suspensions were prepared as described above. Cells were washed with FCM buffer (HBSS containing 1% FBS, 1 mM EDTA, and 10 mM HEPES) and stained with antibodies (Supplementary Table 1) at 4°C in the dark for 20 min. Dead cells were depleted by DAPI staining.
For the immunophenotyping of tumours, tissues were mechanically minced with scissors, and then digested in DMEM containing 1 mg/mL Collagenase IV (Gibco), 100 μg/mL DNase I (Roche), 1% FBS, 10 mM HEPES, 2% Antibiotic-Antimycotic (ThermoFisher Scientific) for 40 min at 37°C in the dark with gentle agitation every 10 min. Digested tissues were then washed twice in DMEM containing 10% FBS, filtered through a 40-μm nylon mesh strainer (Corning). Cells were suspended in ACK Lysing Buffer (ThermoFisher Scientific), incubated for 10 min at 4°C in the dark to remove red blood cells. Cells were washed twice in FCM buffer and counted. Cells were stained with Zombie Aqua Fixable Viability Kit (BioLegend) and blocked with anti-mouse CD16/CD32 antibody (Mouse BD Fc Block™, clone 2.4G2, BD Bioscience). One million cells were incubated with appropriate antibodies (Supplementary Table 1) diluted in FCM buffer at 4°C in the dark for 40 min. Cells were then washed twice with FCM buffer and analyzed or further fixed in 2% PL solution (phosphate-buffered saline (PBS) containing 0.1 M L-Lysine (Sigma) and 2% paraformaldehyde (Ted Pella))53 at 4°C in the dark overnight. Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells were analyzed on a BD LSR Fortessa or a BD LSR-II UV and analyzed by FlowJo software (FlowJo, LLC, version 10.4). Gating strategies were described in Extended Data Fig. 7k and Supplementary Table 2.
Cell sorting
Mouse PDAC cells (HY15549) expressing the GFP-LC3-RFP reporter were grown as organoids for 8 days, harvested and dissociated into single cells as mentioned above. Cells were sorted on MoFlo XDP (Beckman Coulter) based on the GFP/RFP ratio as described in detail elsewhere54,55. The purity of cells after sorting was confirmed by post-sort analyses and was usually > 90%.
Histology and immunohistochemistry
For formalin fixed paraffin-embedded (FFPE) sections, tissues were fixed in 10% buffered formalin, and embedded in paraffin. 5-μm-thick FFPE sections were used for H&E staining. For immunohistochemical staining, FFPE sections were deparaffinized, rehydrated, and incubated in boiling 10 mM pH 6.0 citrate buffer for 20 min for antigen retrieval. To visualize the GFP-LC3-RFP reporter signal or mStrawberry/mSt-Atg4BC74A expression in PDAC cells, tissues were fixed in 2% PL solution at 4°C overnight, incubated in PBS containing 30% sucrose, and embedded in Tissu-Tek OCT compound (Sakura Finetek) on dry ice53. For immunohistochemical staining, 7-μm frozen sections were washed with Tris-buffered saline (pH 7.4) containing 0.05 % Tween-20 (TBS-T), blocked with TBS-T containing 5% goat serum for 1hr at room temperature (RT), and stained with primary antibodies (Supplementary Table 1) at 4°C overnight, followed by incubation with fluorescent-conjugated secondary antibodies (1:400, Supplementary Table 1) at RT for 1 hr. Sections were counter stained with 4 μg/mL Hoechst 33342 (Thermo Fisher) and mounted in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, P36970). Fluorescent images were obtained with Leica DM6 and analyzed using LAS X software (version 2.0.0.14332.2). Bright light images were obtained with a Leica DM2000 bright-field microscope.
For the measurement of GFP-LC3/RFP signal ratio, frozen sections as prepared above were washed with TBS-T 3 times and coverslips were mounted with ProLong™ Diamond Antifade Mountant. Slides were dried at 4°C overnight. Fluorescence images were obtained from at least 4 random fields/tumour with a 20x objective lens. Gray scale, raw-image files (16-bit) were analyzed using ImageJ Software to obtain mean intensities of GFP and RFP signals.
Primary human PDAC specimens (Supplementary Table 3) were generated under Institutional Review Board (IRB)-approved protocol 18–25787 at UCSF. Sections were co-stained with antibodies against CK-19 (1:300) and HLA-A,B (1:300) (Supplementary Table 1). An average of 28 ducts per sample from 9 patient specimens were imaged based on CK-19 expression to ensure analysis of tumour epithelia. The corresponding MHC-I localization per CK-19 positive ductal structure was classified as intracellular if greater than 50% of the staining displayed a non-membrane, punctate distribution.
Quantitative reverse-transcription PCR
Total RNA was extracted using TRIzol (ThermoFisher Scientific) and PureLink RNA Mini Kit (ThermoFisher Scientific), and then reversed transcribed using Superscript Vilo IV (ThermoFisher Scientific) with oligo-dT primers. Quantitative PCR was performed with SYBR Green Supermix (Bio-rad) on the CFX96 real-time PCR machine (Bio-Rad). The quantity of mRNA was calculated using ΔCT method and normalized by the GAPDH, Actb or 36B4 genes. Sequences for qPCR primers are listed in Supplementary Table 4.
Transcriptome analysis
RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA library preparation kit according to manufacturer’s instructions by the NYU Genome Technology Center. The libraries were pooled and sequenced as 50-base, single-end reads on an Illumina HiSeq 4000 using high output mode (v4 chemistry). The raw fastq reads were aligned to mm10 mouse reference genome using STAR aligner56. Fastq Screen was used to check for any contaminations in the samples and Picard RnaSeqMetrics was used to obtain the metrics of all aligned RNA-Seq reads. featureCounts was used to quantify the gene expression levels57. FPKM (fragments per kilobase of transcript per million fragments mapped) data were used as input for gene set enrichment analysis (GSEA)58.
Immunoblotting
Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, #9803) supplemented with protease inhibitor (Thermo Fisher Scientific, # A32953) and phosphatase inhibitor (PhosSTOP, Sigma Aldrich, #04906837001). Proteins were separated on 12% or 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels by SDS PAGE electrophoresis and transferred to onto PVDF membranes (IPVH00010, EMD Millipore) or nitrocellulose membranes (GE Healthcare Amersham™ Protran™ NC Rolls, Fisher, #10600000). Membranes were blocked in 5% non-fat dry milk (Blotting-Grade Blocker, BioRad, #1706404) dissolved in TBS-T for 1 hr and incubated overnight at 4 °C with primary antibodies (Supplementary Table 1). Membranes were washed three times with TBS-T and incubated with secondary antibodies for 1 hr at room temperature: anti-rabbit IgG, HRP-linked (Cell Signaling Technology, #7074), anti-mouse IgG, HRP-linked (Cell Signaling Technology, #7076). Images were obtained by chemiluminescence (Bio-rad 1705061) using a ChemiDoc (Bio-Rad).
Statistical analysis
Statistical analysis was performed using Graphpad Prism 7.0 (GraphPad). Results are expressed as mean +/− standard deviation unless otherwise indicated. For each box-and-whisker plot, centerline is the median and whiskers represent the minimum and maximum values.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
Data availability
RNA-seq data are deposited to the Gene Expression Omnibus (GEO) data repository with accession number GSE145766. Source Data are provided for all experiments. Other data that support the findings of this study are available on request from the corresponding author upon reasonable request.
Extended Data
Supplementary Material
Acknowledgements
This work was supported by National Cancer Institute Grants R01CA157490, R01CA188048, P01CA117969, R35CA232124; ACS Research Scholar Grant RSG-13-298-01-TBG; NIH grant R01GM095567; and the Lustgarten Foundation, and SU2C to A.C.K. R.M.P is the Nadia’s Gift Foundation Innovator of the Damon Runyon Cancer Research Foundation (DRR-46-17) and is additionally supported by an NIH Director’s New Innovator Award (1DP2CA216364) and the Pancreatic Cancer Action Network Career Development Award. K.Y was supported by the Uehara Memorial Foundation Research Fellowship. A.V is supported by a National Science Foundation Graduate Research Fellowship. D.E.B. is supported by a Ruth L. Kirschstein Institutional National Research Service Award, T32 CA009161 (Levy), and the NCI Predoctoral to Postdoctoral Fellow Transition Award (F99/K00) F99 CA245822. M.K. was supported by the Uehara Memorial Foundation Research Fellowship and Postdoctoral Fellowship for Research Abroad (Japan Society for the Promotion of Science). S.J.P. was supported by American Cancer Society grant 132942-PF-18-215-01-TBG. R.S.B. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2348-18).
We thank Haoqiang Ying for providing the HY19636 and HY15549 cells. We thank the New York University (NYU) Langone Health Experimental Pathology Laboratory, Flow Cytometry Core, and Genome Technology Center, each supported in part by the Cancer Center Support grant P30CA016087 at the Laura and Isaac Perlmutter Cancer Center. We thank Zahid Dewan for the technical assists on the staining of frozen sections. We acknowledge the UCSF Parnassus Flow Cytometry Core (PFCC) supported in part by the DRC Center Grant P30DK063720 for assistance generating flow cytometry data. We thank Dr. James Olzmann (UC Berkeley) for advice on ubiquitylation experiments. We thank all the members of Perera lab, Kimmelman lab, Debnath Lab and Pacold lab for helpful suggestions.
Footnotes
Competing interests
A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, and the autophagic control of iron metabolism. A.C.K. is on the SAB of Rafael/Cornerstone Pharmaceuticals. A.C.K. is a consultant for Deciphera. J.D. is on the Scientific Advisory Board of Vescor Therapeutics, LLC. The other authors declare no competing interests.
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Associated Data
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Supplementary Materials
Data Availability Statement
RNA-seq data are deposited to the Gene Expression Omnibus (GEO) data repository with accession number GSE145766. Source Data are provided for all experiments. Other data that support the findings of this study are available on request from the corresponding author upon reasonable request.