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Sup Tables 5-7- Amino Acid Biochrom

TMA Clinical Data

RNAseq

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.Significance:This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211

Amino Acid- Mass Spec

Microarray

TMA IHC Scores

18 supplementary figures and corresponding legends

Lists all key resources and reagents

PDF file - 1.2MB, Fig. S1. Validation of the sterol pathway targeting siRNA. Fig. S2. Silencing of SC4MOL increased erlotinib-induced apoptosis. Fig. S3. Metabolic effects of SC4MOL silencing and sterols supplementation. Fig. S4. Sterol pathway interactions. Fig. S5. Effects of SC4MOL silencing on EGFR synthesis and I-125 EGF internalization and degradation. Fig. S6. Effects of sterol pathway genes silencing on EGFR endosomal traffic. Fig. S7. Effects of SC4MOL silencing on EGFR ubiquitin conjugation and accumulation in late endosomes. Fig. S8. Supporting data for Figure 5. Fig. S9. Supporting data for Figure 7A. Fig. S10. Analysis of NSDHL expression in malignant and benign epithelial tissues

PDF file - 105K

Supplemental Figure 4. mRNA abundance in triptolide-treated PDAC 001 cells

Purpose: Even when diagnosed prior to metastasis, pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with almost 90% lethality, emphasizing the need for new therapies optimally targeting the tumors of individual patients.Experimental Design: We first developed a panel of new physiologic models for study of PDAC, expanding surgical PDAC tumor samples in culture using short-term culture and conditional reprogramming with the Rho kinase inhibitor Y-27632, and creating matched patient-derived xenografts (PDX). These were evaluated for sensitivity to a large panel of clinical agents, and promising leads further evaluated mechanistically.Results: Only a small minority of tested agents was cytotoxic in minimally passaged PDAC cultures in vitro. Drugs interfering with protein turnover and transcription were among most cytotoxic. Among transcriptional repressors, triptolide, a covalent inhibitor of ERCC3, was most consistently effective in vitro and in vivo causing prolonged complete regression in multiple PDX models resistant to standard PDAC therapies. Importantly, triptolide showed superior activity in MYC-amplified PDX models and elicited rapid and profound depletion of the oncoprotein MYC, a transcriptional regulator. Expression of ERCC3 and MYC was interdependent in PDACs, and acquired resistance to triptolide depended on elevated ERCC3 and MYC expression. The Cancer Genome Atlas analysis indicates ERCC3 expression predicts poor prognosis, particularly in CDKN2A-null, highly proliferative tumors.Conclusions: This provides initial preclinical evidence for an essential role of MYC–ERCC3 interactions in PDAC, and suggests a new mechanistic approach for disruption of critical survival signaling in MYC-dependent cancers. Clin Cancer Res; 22(24); 6153–63. ©2016 AACR.

AUC results of the drug sensitivity screens in conditionally reprogrammed PDAC cells.

Table S1. Genetic characterization of patient-derived xenografts using the Illumina Trusight panel capturing frequent mutations in 94 genes and 284 SNPs associated with cancer Table S3. Triptolide-induced effects on the mRNA abundance was determined by Illumina HighSeq RNA sequencing of 001 cells treated with 100 nM triptolide in vitro for 6 hours.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. Significance: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230. This article is highlighted in the In This Issue feature, p. 211

BackgroundEngineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but solid tumors remain an intractable challenge. We have previously developed a human chimeric antigen receptor macrophage (CAR-M) platform for adoptive cell therapy and shown potent anti-tumor activity in pre-clinical solid tumor models.1 CAR-M overcome critical solid tumor challenges such as tumor infiltration, immunosuppression within the tumor microenvironment, lymphocyte exclusion, and target antigen heterogeneity. Currently, CAR-M are generated in a week-long ex-vivo process in which peripheral blood monocytes are differentiated into macrophages prior to genetic manipulation. Here, we demonstrate the production feasibility, phenotype, pharmacokinetics, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes.MethodsUsing the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR targeted against human epidermal growth factor receptor 2 (HER2) (CAR-mono). Using a partially automated approach, we established a process that allowed for same day manufacturing (from Leukopak to cryopreserved CAR-mono cell product).ResultsCAR expression and cell viability exceeded 90%, and cells efficiently differentiated into CAR-expressing macrophages. The adenoviral based gene modification method led to pre-conditioning of CAR-mono cells resulting in a strong M1 phenotype upon differentiation, and potent anti-tumor activity regardless of exposure to GM-CSF, M-CSF, or immunosuppressive factors. Treating CAR-mono cells with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs, indicating that these cells retain their myeloid differentiation potential. In vivo, CAR-mono treatment induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following intravenous administration, CAR-mono demonstrated the ability to traffic to both GM-CSF < sup >high and GM-CSF< sup >lowexpressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (>100 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support.ConclusionsThe CAR-mono platform allows for a rapid, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. Ad5f35 engineered human monocytes are primed toward M1 macrophage differentiation and produce a cell population highly similar to our established CAR-M platform. Collectively, these findings provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.ReferenceKlichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology March 2020.

BackgroundDespite the remarkable efficacy achieved by CAR-T therapy in hematologic malignancies, application in solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival.1 Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the TME and the adaptive immune system.MethodsMurine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic CAR-M monotherapy or in combination with a PD1 blocking antibody. Tumors were collected at various time points and dynamic changes in the TME were assessed using flow cytometry, immunohistochemistry, and gene expression analysis.ResultsIn addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. In vivo, CAR-M led to significant tumor regression and improved overall survival in multiple syngeneic models. Analysis of the TME showed that CAR-M led to increased infiltration of intratumoral CD4+ and CD8+ T, NK, and dendritic cells – as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further reprogrammed the TME, enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of any serum analyte associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessmentsConclusionsThese results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors for the treatment of solid tumors.ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953

Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). Trogocytosis is a process of “nibbling” of plasma membranes between two living cells. Here, we demonstrate CAFs are the primary recipients of exogenous lipids which they transfer to metabolically “parasitic” PDAC cells via a contact-mediated trogocytosis. Whereas trogocytosis has been described in immune system and in normal development, the biochemical and signaling regulators of trogocytosis between CAFs and PDAC cells have not been defined. Results: We determine that trogocytosis occurs through heterotypic CAF-PDAC cell contacts: membrane blebbing (readily observed in activated CAFs and exaggerated by release of Ca2+ from the ER stores) results in the blebs uptake by the PDAC cells. The CAF blebs express externalized phosphatidylserine (PtdSer), and blockade of PtdSer in vitro transiently deters trogocytic uptake of CAF membranes. Probing a short list of candidate targets involved in regulation of cholesterol trafficking, membrane fusion and membrane protrusions using CRISPRi, has shown that CAFs deficient in CD81, ARF6 or TMEM16F exhibit markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing PtdSer on the outer leaflet of the plasma membrane. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures, and do not support the growth of PDAC cells in orthotopic co-implantation model. Furthermore, several clinically available antibiotics used to treat tapeworms are selective TMEM16F inhibitors. One of the widely available TMEM16F inhibitors, niclozamide, is effective in blocking cholesterol transfer from CAFs to PDAC cells in vivo. Conclusion: Our overall model is that activated CAFs initiate trogocytosis by expressing PtdSer as “eat me” signals on the surfaces of their membrane blebs. This process is regulated by Ca2+-dependent phospholipid scramblase TMEM16F which is an attractive drug-amenable target to dismantle the critical metabolic dependency in PDAC on the exogenous lipids. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova-Cornell, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Debora Barbosa Vendramini-Costa, Ralph Francescone, Janusz Franco-Barraza, Jaye Gardiner, Emmanuelle Nicolas, Andrei Efimov, Elizabeth A. Handorf, Kathy Q. Cai, Bojana Gligorijevic, Edna Cukierman, Igor Astsaturov. Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C047.

Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide nutrients to "parasitic" PDAC cells including water soluble glucose and amino acids. In hypoxic tumor microenvironments, aggressive PDAC are functional auxotroph for lipids, and scavenge exogenous lipids to build cellular membranes. However, the mechanism by which PDAC cells obtain these water-insoluble essential membrane building materials remains poorly understood. To gain insights into the mechanism of lipid scavenging, we generated cholesterol-auxotrophic human and mouse PDAC PDAC cells since cholesterol is the major constituent of lipid cellular membranes. Here, we discovered that PDAC cells utilize CAFs as a main source of lipids in vivo through direct heterotypic cellular contacts. In this process, PDAC cells directly acquire the CAF plasma membrane (PM) via trogocytosis. In response to yet unidentified paracrine “feed me” signals activating CAF-dependent trogocytosis, CAFs upregulate phosphatidylserine (PtdSer) on the outer leaflet of the PM. Consequently, blockade of PtdSer on CAFs partially deterred trogocytic transfer of CAF membranes to PDAC cells in vitro. Furthermore, Ca2+-dependent phospholipid scramblase TMEM16F is a critical regulator of PtdSer externalization. TMEM16F is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the matching adjacent non-malignant pancreatic tissues CAFs deficient in TMEM16F scramblase exhibited markedly reduced ability to support the growth of cholesterol-auxotrophic PDAC cells cultured in lipid depleted media. We propose trogocytosis as a new mode of lipid scavenging by PDAC cells from CAFs involving activation of Ca2+-dependent phospholipid scramblase TMEM16F in CAFs and increased expression of PtdSer as “eat me” signals on CAF PM. To inactivate trogocytosis, we nominate TMEM16F as a plausible PDAC therapy target using clinically available TMEM16 inhibitors with a potential for impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Battuya Bayarmagnai, Linara Gabitova, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Debora Vendramini Costa, Ralph Francescone, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1571.

Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, achieving efficacy against solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the endogenous immune system and to study the combinatorial approach of CAR-M with blockade of the PD1/PDL1 T cell checkpoint axis. Murine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. In addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. Anti-HER2 CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic HER2-CAR or untransduced (UTD) macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in tumors resistant to anti-PD1 monotherapy and found that the combination further reprogrammed the TME, significantly enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2-negative rechallenge model, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of serum analytes associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessments. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Alison Worth, Ilyssa Ramos, Daniel Blumenthal, Yumi Ohtani, Linara Gabitova, Michael Ball, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2112.

Introduction: Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but responses have been rare in solid tumors. Our previously developed human chimeric antigen receptor macrophage (CAR-M) platform has shown potent anti-tumor activity in pre-clinical solid tumor models1, and the anti-HER2 CAR-M CT-0508 is currently being evaluated in a Phase I trial. The use of myeloid cells as a platform for cell therapy provides the tools to overcome critical solid tumor challenges such as infiltration, immunosuppression within the tumor microenvironment, lymphocyte exclusion, and target antigen heterogeneity. Currently, CAR-M are generated in a week-long ex-vivo process in which peripheral blood monocytes are differentiated into macrophages prior to genetic manipulation. Here, we demonstrate the production feasibility, phenotype, pharmacokinetics, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes. Experimental: Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR (CAR-mono) targeted against HER2. We established a process that allowed for same day manufacturing (from Leukopak to cryopreserved CAR-mono cell product). Results: CAR-mono showed high CAR expression and viability (>90%), and efficiently differentiated into CAR-expressing macrophages. Adenoviral transduction led to pre-conditioning of CAR-mono, resulting in a strong M1 phenotype upon differentiation into CAR-M. CAR-mono derived macrophages demonstrated potent anti-tumor activity regardless of exposure to GM-CSF or M-CSF, and were protected against M2 switching by immunosuppressive factors. Treating CAR-mono with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs with an activated phenotype, indicating that these cells retained their myeloid differentiation potential. In vivo, CAR-mono induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following IV administration, CAR-mono demonstrated the ability to traffic to both GM-CSFhigh and GM-CSFlow expressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (>180 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support. Conclusions: The CAR-mono platform enables an automated, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. The use of Ad5f35 for human monocyte transduction primes the cells toward M1 macrophage differentiation and produces a cell population phenotypically and functionally similar to our established CAR-M platform. These data provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.1Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. March 2020. Citation Format: Daniel Blumenthal, Linara Gabitova, Brett Menchel, Patricia Reyes-Uribe, Sabrina Ceeraz DeLong, Sascha Abramson, Michael Klichinsky. Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 582.

Gene expression analyses established at least two molecular subtypes of pancreatic adenocarcinoma (PDAC), the classical (or glandular), and the basal (or mesenchymal), each of which is associated with distinct prognoses and sensitivity to chemotherapy. It remains unclear, however, whether the basal carcinoma cells arise from a separate cell-of-origin, or are emerging from the pre-existing well-differentiated “classical” PDAC cells. To distinguish these alternatives, we conducted single-cell transcriptome analyses and virtual lineage tracing comparing cellular populations at pre-malignant stages in basal versus classical PDAC mouse models. We previously reported that chemical or genetic inhibition of the cholesterol biosynthetic pathway in KrasG12D; Trp53 (KPPC) mice predisposes to basal rather than glandular PDAC development because of the pancreas-specific increased sterol response element-binding protein 1 (SREBP1) activity and TGFβ signaling that induces cancer cell stemness and the EMT (PMID: 32976774). Pancreas-selective knockout of a conditional allele of cholesterol pathway gene, Nsdhl (NAD(P)-dependent steroid dehydrogenase-like), renders pancreatic epithelial cells cholesterol auxotrophs and drives basal PDAC in the majority of animals (KPPCN mice). At 5-6 weeks of age, grossly and microscopically tumor-free pancreatic tissues were selected for single-cell isolation and single-cell RNA sequencing (scRNA seq) using the 10X platform. After standard filtering and sample normalization procedures, downstream analyses included identification of relevant cell clusters using Seurat, lineage tracing algorithms, and in silico modeling of autocrine and paracrine signaling interactions between subsets of PDAC and non-malignant cells. Our key findings are as follows: 1) premalignant KPPCN pancreata exhibit a massive expansion of cancer-associated fibroblasts (CAFs) of predominantly inflammatory differentiation (iCAFs); 2) despite relatively fewer ADM and PanIN pre-malignant lesions in KPPCN compared to KPPC, scRNA seq identifies the significant expansion of epithelial cells with features of centroacinar and stem-like cells (increased expression of Aldh1a2, Nes, Sox9, Ly6a, Cxcl12, and Met); these centroacinar-like cells, while retaining epithelial identity (Epcam, Cdh1), also exhibit features of pluripotency by co-expression of Ins2 and other stem cell markers; 3) alignment with basal PDAC (KPPCN) and classical (KPPC) carcinoma cell populations strongly suggests the continuity of clonal evolution of the centroacinar-like cells towards the basal PDAC. While our genetic model does not recapitulate the multiple alternative pathways leading to basal PDAC development, cholesterol auxotrophy via SREBP1 may be a factor governing the expansion of undifferentiated precursors, which via interactions with cancer-promoting iCAFs, drive basal PDAC development. Citation Format: Michael Kotliar, Aizhan Surumbayeva, Linara Gabitova, Suraj Peri, Diana Restifo, Kathy Q. Cai, Artem Barski, Igor Astsaturov. Cholesterol auxotrophy promotes the expansion of centroacinar cells giving rise to the basal subtype of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-068.

Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). CAFs have been shown to provide to metabolically “parasitic” PDAC cells with water-soluble metabolites such as glucose and amino acids, or bulk protein via micropinocytosis. To meet the elevated demands for cellular membrane lipids, cancer cells rely on uptake of the exogenous lipids. However, the mechanism by which PDAC cells obtain water-insoluble essential lipids, such as cholesterol, remains poorly understood. Here, we define CAFs as a main source of lipids for PDAC cells by direct “feeding” of the CAF plasma membrane (PM) to cancer cells via heterotypic cellular contacts, a phenomenon known as trogocytosis. To gain insights into the mechanisms of regulation of CAF trogocytosis, we engineered cholesterol-auxotrophic human and mouse PDAC cells. In the absence of exogenously provided cholesterol, these cancer cells undergo apoptosis, which is completely rescued in CAF co-cultures. Using CRISPRi-mediated depletion in CAFs of select genes involved in cholesterol trafficking, membrane fusion and membrane protrusions, we found that CAFs deficient in CD81, TMEM16F, or ARF6 exhibited markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing phosphatidylserine (PtdSer) on the outer leaflet of the PM. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures. Our overall model is that, to initiate trogocytosis, PDAC cells release paracrine “feed me” signals activating Ca2+-dependent phospholipid scramblase TMEM16F. As the result, increased outward expression of PtdSer on CAF PM is a hallmark “eat me” signal that is recognized by the trogocytic PDAC cells. We conclude that trogocytosis is the critical metabolic dependency in PDAC, and nominated TMEM16F as a plausible PDAC therapy target. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Janusz Franco-Barraza, Ralph Francescone, Debora B. Barbosa Vendramini-Costa, Jaye Gardiner, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Edna Cukierman, Igor Astsaturov. Cancer-associated fibroblasts sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-015.

Recent advances in cell therapy have led to significant efficacy in hematologic malignancies, but solid tumors remain an intractable challenge. We have previously developed a CAR macrophage (CAR-M) adoptive cell therapy platform and demonstrated potent anti-tumor activity in pre-clinical models. CAR-M overcome several of the barriers to efficacy in the solid tumor setting - trafficking, immunosuppression, lymphocyte exclusion, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. In order to streamline the manufacturing process, improve cell yields, and potentially improve tumor infiltration, we sought to evaluate the feasibility of directly engineering CD14+ monocytes to express CAR. Using the chimeric adenoviral vector Ad5f35, we engineered primary human CAR-monocytes to target tumors overexpressing HER2. CAR expression and viability both exceeded 90%. Anti-HER2 CAR-monocytes produced pro-inflammatory cytokines in response to antigen, specifically phagocytosed HER2 overexpressing target cells, and eradicated HER2-overexpressing tumor cells in a time and dose-dependent manner. CAR-monocytes efficiently differentiated into CAR-expressing macrophages in response to various maturation factors and cytokines within 3-5 days. Adenoviral based gene modification resulted in the upregulation of several pro-inflammatory markers on CAR-monocytes and pre-conditioned these cells to differentiate into M1 macrophages in the absence of exogenous M1 polarization factors. The M1 phenotype was maintained when CAR-monocytes were challenged with immunosuppressive cytokines in vitro. These cells demonstrated potent effector function after differentiation into macrophages, regardless of exposure to GM-CSF, M-CSF, or immunosuppressive factors. Anti-HER2 CAR monocytes carried broadly expressed myeloid chemokine receptors and responded to a panel of chemotactic factors. In order to optimize the cell manufacturing process associated with CAR-M, we established a closed-system, ultra-rapid CAR monocyte process. This process enabled same day manufacturing of the final CAR monocyte cell product, significantly reducing the cost of goods associated with autologous cell therapy as well as “vein to vein” time. This study demonstrated the successful development of CAR-monocytes with direct anti-tumor activity and capacity to differentiate into M1 polarized CAR-M. In addition, we established an ultra-rapid same-day CAR monocyte manufacturing process. The CAR-monocyte platform represents an advance in the field of adoptive cell therapy. Citation Format: Linara Gabitova, Brett Menchel, Rashid Gabbasov, Stefano Pierini, Andrew Best, Kayleigh Ross, Yumi Ohtani, Daniel Blumenthal, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1530.

Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, translating these results in solid tumors remains challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Herein, we established a fully immunocompetent syngeneic mouse model and evaluated the interaction of CAR-M with the tumor microenvironment (TME) and the endogenous adaptive immune system. Murine bone marrow-derived CAR-expressing macrophages (muCAR-M) were efficiently engineered to express an anti-huHER2 CAR using the chimeric adenoviral vector Ad5f35. MuCAR-M, but not control untransduced (UTD) macrophages, specifically phagocytosed HER2+ target cancer cell lines and killed HER2-expressing murine CT26 colorectal and human AU-565 (HER2+) breast cancer cells in a dose-dependent manner. Moreover, CAR-M induced MHC-I and MHC-II expression on tumor cells and cross-presented tumor-associated antigens (TAA) resulting in CD8 T cell activation. To evaluate muCAR-M in an immunocompetent in vivo setting, BALB/c mice were engrafted with subcutaneous CT26-HER2+ tumors and treated with HER2-CAR or UTD macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cells reactive to the CT26 MHC-I antigen gp70, indicating enhanced epitope spreading. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2- CT26 rechallenge model, indicating the induction of long-term T cell memory against TAA. To evaluate the systemic anti-tumor immune response, we simultaneously engrafted BALB/c mice with CT26-HER2+ and CT26-Wt tumors on opposite flanks and treated mice with local administration of CAR-M restricted to the HER2+ tumors. After CAR-M treatment, 75% of mice cleared their CT26-HER2+ tumors and the growth rate of the contralateral CT26-Wt tumors was significantly reduced, demonstrating an abscopal effect. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with PD1 checkpoint inhibitor therapy in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further improved tumor control and overall survival. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Linara Gabitova, Yumi Ohtani, Olga Shestova, Saar Gill, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 63.

SummaryOncogenic transformation alters the metabolism of cellular nutrients to sustain tumor growth. We here define a mechanism by which modifications in cholesterol metabolism control the formation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by means of conditional inactivation ofNsdhlin mice bearing a tumor-inducingKrasmutation (KrasG12D) prevented PDAC formation in the context of a heterozygousTrp53f/+genotype without impairing normal pancreatic development. In mice with pancreaticNsdhlablation and homozygous loss ofTrp53, the emerging tumors presented with the aggressive basal (mesenchymal) phenotype as opposed to the classic (glandular) PDAC. This paralleled significantly reduced expression of cholesterol metabolic pathway genes in human basal PDAC subtype. Mechanistically, we demonstrate that genetic or metabolic cholesterol deprivation stabilizes the transforming growth factor beta (TGFβ) receptor to activate pro-mesenchymal effectors in human and murine PDAC, providing a direct mechanism by which cholesterol metabolism can condition tumor differentiation.

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by KrasG12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor β signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.

Despite rapid advances in cancer immunotherapy, clinical responses in metastatic solid tumors have been limited. Macrophages are the most abundant immune cell in the solid tumor microenvironment (TME) and are primarily recruited as monocytes by TME-derived chemokines. When not under the control of the TME, macrophages are potent immune effector cells capable of phagocytosis, T cell recruitment, and antigen presentation. We have previously demonstrated that CAR macrophages (CAR-M) have potent anti-tumor activity and overcome several of the barriers to success in solid tumor immunotherapy - trafficking, immunosuppression, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. To more closely recapitulate normal biologic behavior, we attempted to create CAR monocytes that could traffic and differentiate into CAR macrophages upon tumor penetration. Toward that goal, we genetically engineered CD14+ human monocytes without ex vivo differentiation and with minimal cell culture. Using the chimeric adenoviral vector Ad5f35, we engineered human CAR-monocytes targeted against HER2. CAR expression and viability both exceeded 90%. Ad5f35 transduced CAR monocytes survived and maintained CAR expression ex vivo for at least 21 days. CAR monocytes efficiently differentiated into CAR-expressing macrophages when treated with GM-CSF as determined by FACS-based phenotypic characterization and Wright-Giemsa staining. Anti-HER2 CAR monocytes eradicated HER2 expressing tumor cells in a time and dose-dependent manner, and had comparable potency to anti-HER2 CAR-M. Additionally, the CAR monocyte manufacturing process offered the logistical advantage of a short manufacturing process (approximately two days). We have previously demonstrated that CAR-M are polarized toward a pro-inflammatory M1 phenotype after transduction with Ad5f35. Similarly, CAR monocytes demonstrated elevated expression of M1 markers, and intriguingly after differentiation into CAR-expressing macrophages, HLA-DR, CD80, CD86, and other M1 markers remained elevated - suggesting that transduction prior to differentiation does not impact the pro-inflammatory impact of adenoviral vectors on myeloid cells. Taken together, this abstract describes the successful development of CAR-monocytes with the potential for a rapid manufacturing process. In addition to direct anti-tumor activity while in the monocyte phase, CAR monocytes have the capacity to differentiate into CAR macrophages in situ, which are in turn capable of phagocytosis, T cell recruitment, TME activation, and antigen presentation. Given the previously demonstrated pre-clinical efficacy of CT-0508 (an anti-HER2 CAR macrophage), the CAR monocyte platform described herein offers a shortened manufacturing process and a potential advantage in tumor penetration, which will be directly evaluated in upcoming studies. Citation Format: Konrad Gabrusiewicz, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Linara Gabitova, Daniel Cushing, Saar Gill, Michael Klichinsky. Genetically engineered chimeric antigen receptor (CAR) monocytes demonstrate targeted anti-tumor activity and differentiate into M1-polarized CAR macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2180.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+ cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+ CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stunted in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.SignificancePDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1 in vivo by limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.

SummaryMeiosis-activating sterols (MAS) are substrates of SC4MOL and NSDHL in the cholesterol pathway and are important for normal organismal development. Oncogenic transformation by epidermal growth factor receptor (EGFR) or RAS increases the demand for cholesterol, suggesting a possibility for metabolic interference. To test this idea in vivo, we ablated Nsdhl in adult keratinocytes expressing KRASG12D. Strikingly, Nsdhl inactivation antagonized the growth of skin tumors while having little effect on normal skin. Loss of Nsdhl induced the expression of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, reduced the expression of low-density lipoprotein receptor (LDLR), decreased intracellular cholesterol, and was dependent on the liver X receptor (LXR) α. Importantly, EGFR signaling opposed LXRα effects on cholesterol homeostasis, whereas an EGFR inhibitor synergized with LXRα agonists in killing cancer cells. Inhibition of SC4MOL or NSDHL, or activation of LXRα by sterol metabolites, can be an effective strategy against carcinomas with activated EGFR-KRAS signaling.

Oncogenic transformation alters the metabolism of cellular nutrients to sustain tumor growth. We here define a mechanism by which modifications in cholesterol metabolism control the formation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by means of conditional inactivation of Nsdhl in mice bearing a tumor-inducing Kras mutation (KrasG12D) prevented PDAC formation in the context of a heterozygous Trp53f/+ genotype without impairing normal pancreatic development. In mice with pancreatic Nsdhl ablation and homozygous loss of Trp53, the emerging tumors presented with the aggressive basal (mesenchymal) phenotype as opposed to the classic (glandular) PDAC. This paralleled significantly reduced expression of cholesterol metabolic pathway genes in human basal PDAC subtype. Mechanistically, we demonstrate that genetic or metabolic reduction in cellular cholesterol induces the expression of transforming growth factor beta (TGF) and activates pro-mesenchymal effectors in human and murine PDAC, providing a direct transcriptional mechanism by which cholesterol metabolism can condition tumor differentiation. Activity of TGF beta pathway estimated via pSMAD2 expression in surgical PDAC samples inversely correlated with patients’ blood cholesterol levels, especially in patients taking cholesterol-lowering statins. Conclusions: Low levels of cellular cholesterol induce autocrine TGF beta signaling in PDAC. Malnutrition or cholesterol-lowering statins may be mechanistically linked to basal PDAC differentiation. Citation Format: Linara Gabitova, Suraj Peri, Ralph Francescone, Janusz Franco-Barraza, Neelima Shah, Emmanuelle Nicolas, Elizabeth A. Handorf, Kathy Q. Cai, Ido Sloma, Rachel Chiaverelli, Richard Moffitt, Erica A. Golemis, Carolyn Fang, Shannon Lynch, Edna Cukierman, Igor Astsaturov. Cholesterol deprivation induces TGFβ signaling to promote basal differentiation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C06.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to soon become the second killer cancer in the USA. One of the hallmarks of PDAC is desmoplasia, which consists of an expansion in activated stellate cells, known as cancer associated fibroblasts (CAFs), and remodeling of their extracellular matrix. Desmoplasia is a major culprit for the unique PDAC microenvironment, which both collapses blood vessels restricting nutrients and fosters immunosuppression. Crosstalk between CAFs, PDAC, and immune cells, within this microenvironment, is not fully understood. To study the functions of CAFs on PDAC development, we first performed a microarray comparing patient matched normal pancreatic stellate cells and CAFs cultured using our stromal 3D system. We identified proteins upregulated in CAFs that have not been previously implicated in desmoplasia. To understand the functional role of these proteins, CRISPR/Cas9 mediated deletion of target proteins was undertaken and cells were subjected to a number of co-culture assays, with PDAC cells and NK cells. Assays consisted of material transfer, cell engagement, proliferation, survival, NK cell activation and PDAC killing. Syngeneic/orthotopic in vivo models, RNAseq analyzes and quantitative multi-plex microscopy, on pathological samples, were also used. We uncovered the ectopic expressions of NetrinG1 (NG1), a neural pre-synaptic protein, in CAFs, and of NG1’s post-synaptic binding partner, NGL1, in PDAC cells. Using our 3D system, we observed that heterotypic CAF-to-PDAC interactions, via NG1/NGL1 engagement, are critical for providing survival advantages to nutrient deprived PDAC cells and for CAFs to protect PDAC from NK cell-driven elimination. Mechanistically, we uncovered that PDAC starvation is overcome by NG1 expressing CAFs that provide nutrition via material transfer. Further, knockout of NG1, in CAFs, significantly reduced the production of immunosuppressive cytokines, such as IL-6, IL-8, and TGF-β. Moreover, loss of NG1 from CAFs resulted in anti-tumor NK cell activation. Unexpectedly, we observed a link between glutamate metabolism in CAFs and the inactivation of anti-tumor NK cells, and this was dependent on CAFs’ NG1 expression. These results were confirmed in an orthotopic PDAC mouse model in which ablation of NGL1, in syngeneic mouse PDAC cells, significantly halted tumor growth. RNAseq comparing CON or NG1+ CAFs vs. knockout NG1- CAFs rendered changes in neural, metabolic and fibrous signature pathways. Translationally, we saw clear expression of stromal NG1 only in PDAC affected pancreata. Overall, this study identifies two novel targets for PDAC; a cancer that lacks effective therapies. Inhibition of the NG1/NGL1 axis, constitutes a potential future therapeutic approach, as it alters CAF mediated metabolism and reverts immunosuppression, stunting PDAC growth in vitro and in vivo. Citation Format: Ralph Francescone, Débora Barbosa Vendramini-Costa, Janusz Franco-Barraza, Jessica Wagner, Alex Muir, Linara Gabitova, Tatiana Pazina, Tiffany Luong, Neelima Shah, Dustin Rollins, Ruchi Malik, Sapna Gupta, Roshan Thapa, Diana Restifo, Allison Lau, Yan Zhou, Kathy Q. Cai, Harvey Harvey Hensley, Emmanuelle Nicolas, Warren D. Kruger, Karthik Devarajan, Siddharth Balachandran, Wafik S. El-Deiry, Matthew Vander Heiden, Kerry Campbell, Igor Astsaturov, Edna Cukierman. NG1/NGL1 engagement supports PDAC development via CAF to PDAC nutrition and CAF-regulated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2038.

Background: Dual specificity phosphatase 6 (DUSP6) is a member of a family of mitogen-activated protein kinase phosphatases that dephosphorylates and inhibits activated ERK1/2. Dual specificity phosphatase 6 is dynamically regulated in developmental and pathological conditions such as cancer. Methods: Cancer cell lines were made deficient in DUSP6 by siRNA and shRNA silencing. Sensitivity to anti-EGFR and chemotherapeutic agents was determined in viability and apoptosis assays, and in xenografts established in SCID mice. Cellular effects of DUSP6 inactivation were analysed by proteomic methods, followed by analysis of markers of DNA damage response (DDR) and cell cycle. Results: We determined that depletion of DUSP6 reduced the viability of cancer cell lines and increased the cytotoxicity of EGFR and other targeted inhibitors, and cytotoxic agents, in vitro and in vivo. Subsequent phosphoproteomic analysis indicated DUSP6 depletion significantly activated CHEK2 and p38, which function in the DDR pathway, and elevated levels of phosphorylated H2AX, ATM, and CHEK2, for the first time identifying a role for DUSP6 in regulating DDR. Conclusion: Our results provide a novel insight into the DUSP6 function in regulating genomic integrity and sensitivity to chemotherapy in cancer.

Persistent signaling by the oncogenic EGF receptor (EGFR) is a major source of cancer resistance to EGFR targeting. We established that inactivation of 2 sterol biosynthesis pathway genes, SC4MOL (sterol C4-methyl oxidase–like) and its partner, NSDHL (NADP-dependent steroid dehydrogenase–like), sensitized tumor cells to EGFR inhibitors. Bioinformatics modeling of interactions for the sterol pathway genes in eukaryotes allowed us to hypothesize and then extensively validate an unexpected role for SC4MOL and NSDHL in controlling the signaling, vesicular trafficking, and degradation of EGFR and its dimerization partners, ERBB2 and ERBB3. Metabolic block upstream of SC4MOL with ketoconazole or CYP51A1 siRNA rescued cancer cell viability and EGFR degradation. Inactivation of SC4MOL markedly sensitized A431 xenografts to cetuximab, a therapeutic anti-EGFR antibody. Analysis of Nsdhl-deficient Bpa1H/+ mice confirmed dramatic and selective loss of internalized platelet-derived growth factor receptor in fibroblasts, and reduced activation of EGFR and its effectors in regions of skin lacking NSDHL. Significance: This work identifies a critical role for SC4MOL and NSDHL in the regulation of EGFR signaling and endocytic trafficking and suggests novel strategies to increase the potency of EGFR antagonists in tumors. Cancer Discov; 3(1); 96–111. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 1

Pancreatic cancer is rapidly rising to become the 2nd leading cause of cancer deaths by 2020 in the USA. The rise in pancreatic cancer incidence is paralleled by the epidemic of obesity, type 2 diabetes, and associated increase in blood cholesterol, although mechanistic explanations for this are currently unknown. We and others have demonstrated that cancers with activated EGFR and KRAS signaling have increased demands for cholesterol and are vulnerable to interference with cholesterol uptake or endogenous cholesterol biosynthesis. We recently identified a metabolic step in the distal cholesterol pathway, mediated by SC4MOL and NSDHL enzymes, as a critical regulator of cell growth in the keratinocyte model of KRAS-induced carcinogenesis via the LXR agonistic effect of accumulating C4-methylsterols. Based on these preliminary data, we set out to test if pancreatic cancer initiation and progression depend on accelerated cholesterol biosynthesis in a genetic mouse model containing a conditional cholesterol pathway enzyme deficiency (conditional knockout of Nsdhlf/f) in the context of pancreatic cancer development in KPC mice (LSL-KrasG12D;Tp53f/f;Pdx1-Cre). Results: Conditional inactivation of NSDHL alone in pancreatic tissue during normal development produced no apparent phenotype. As expected, the NSDHL-sufficient KPC mice as well as heterozygous Nsdhlf/+ littermates did not survive beyond 8 weeks of age due to rapid development of progressive pancreatic tumors showing complete displacement of pancreatic tissue with adenocarcinoma and high-grade PanIN lesions. Contrastingly, the survival of pancreatic conditional NSDHL-null mice was significantly extended beyond the median survival of 50 days in NSDHL-sufficient age-matched controls. Furthermore, the progression of pancreatic lesions from ADM to PanIN3 was remarkably delayed on NSDHL-null background, with only a proportion of animals developing adenocarcinoma. Evaluation of pancreatic tissues revealed a dramatic reduction of tumor-induced desmoplasia at all stages of pancreatic cancer development. Our ongoing studies will address the role of cholesterol metabolism in pancreatic cancer progression via regulation of tumor-specific production of stroma-recruiting growth factors. Conclusions: Our studies demonstrated for the first time essential genetic evidence for metabolic dependency of pancreatic cancer on cholesterol metabolism. We identified NSDHL as a critical target in the endogenous pathway of cholesterol biosynthesis, and determined that blockade of NSDHL has dramatic consequences on the reciprocal signaling between the KRAS-transformed pancreatic cancer cells and the stroma. Citation Format: Linara Gabitova, Alena Klochkova, Diana Restifo, Aleksandra Mazitova, Edna Cukierman, Tiffiney Hartman, Igor Astsaturov. Cholesterol biosynthesis is a critical metabolic dependency in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2017-438

Accelerated cholesterol and lipid metabolism are the hallmarks of cancer and contribute to malignant transformation due to the obligatory requirement for cholesterol for the function of eukaryotic membranes. To build new membranes and maintain active signaling, cancer cells depend on high intensity of endogenous cholesterol biosynthesis and uptake of lipid particles. This metabolic dependency of cancer cells on cholesterol and other lipids is tightly regulated by the cholesterol homeostasis network, including (i) sterol response element–binding proteins (SREBP), master transcriptional regulators of cholesterol and fatty acid pathway genes; (ii) nuclear sterol receptors (liver X receptors, LXR), which coordinate growth with the availability of cholesterol; and (iii) lipid particle receptors, such as low-density lipid particle (LDL) receptor, providing exogenous sterol and lipids to cancer cells. In addition, activity of oncogenic receptors, such as MUC1 or EGFR, accelerates sterol uptake and biosynthesis. Therefore, a general strategy of reducing the cholesterol pool in cancer cells is challenged by the highly efficient feedback loops compensating for a blockade at a single point in the cholesterol homeostatic network. Besides the well-established structural role of cholesterol in membranes, recent studies have uncovered potent biologic activities of certain cholesterol metabolic precursors and its oxidized derivatives, oxysterols. The former, meiosis-activating sterols, exert effects on trafficking and signaling of oncogenic EGF receptor (EGFR). Cholesterol epoxides, the highly active products of cholesterol oxidation, are being neutralized by the distal sterol pathway enzymes, emopamyl-binding protein (EBP) and dehydrocholesterol-7 reductase (DHCR7). These recently discovered “moonlighting” activities of the cholesterol pathway genes and metabolites expand our understanding of the uniquely conserved roles these sterol molecules play in the regulation of cellular proliferation and in cancer. Clin Cancer Res; 20(1); 28–34. ©2013 AACR.

We identified SC4MOL and NSDHL, two enzymes in the cholesterol pathway and their substrates, meiosis activating sterols (MAS), as critical regulators of receptor signaling and trafficking in normal development and in cancer. Oncogenic transformation by EGFR or RAS increases the demand for cholesterol, suggesting a possibility for metabolic interference. To test this idea in vivo, we ablated Nsdhl in adult keratinocytes expressing KRASG12D. Strikingly, Nsdhl inactivation antagonized the growth of skin tumors, while having little effect on normal skin. Loss of Nsdhl induced the expression of ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, reduced the expression of low-density lipoprotein receptor (LDLR), decreased intracellular cholesterol and was dependent on the liver X receptor (LXR) α. Importantly, EGFR signaling opposed LXRα effects on cholesterol homeostasis, while an EGFR inhibitor synergized with LXRα agonists in killing cancer cells. Inhibition of SC4MOL or NSDHL, or activation of LXRα by sterol metabolites can be an effective strategy against carcinomas with activated EGFR-KRAS signaling. Citation Format: Linara Gabitova, Diana Restifo, Elizabeth Handorf, Kathy Q. Cai, Igor A. Astsaturov. Sensitive step in cholesterol biosynthesis reveals role for sterol metabolites in regulating growth of EGFR/KRAS-dependent tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 26.

Cellular growth is highly dependent on sustained production of lipids. Sterol composition of cellular membranes determines multiple biochemical and biophysical properties of membrane-based processes including vesicle traffic, receptor signaling, and assembly of protein complexes. Lipid biogenesis has become an attractive biochemical target in cancer given the high level of dependency on sterols and lipids in a cancer cell. This review summarized the current knowledge of mechanisms of interaction between the metabolism of sterols and receptor signaling.

Sterols are structural components of lipid membranes which can exert potent biological activities by regulating cell surface receptor trafficking and stability. Arrest of the sterol pathway at the level of SC4MOL (sterol C4-methyl oxydase-like) or NSDHL (NADPH-dependent steroid dehydrogenase-like) specifically antagonizes signaling and trafficking of the epidermal growth factor receptor (EGFR) (Sukhanova, 2013). SC4MOL and NSDHL catalyze two sequential steps of oxidative decarboxylation of the C4-methyl groups of meiosis activating sterols (MAS). The protein binding MAS has not been identified. We investigated whether the anti-EGFR activities of MAS metabolites are mediated via their interaction with the Liver X Receptor (LXR) so that LXR and its transcriptional targets induce subsequent deregulation of intracellular cholesterol homeostasis. In EGFR-positive carcinoma cell, inhibition of SC4MOL or NSDHL induces upregulation of two canonical LXR targets: the ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1). In keeping with the LXR activation, expression of low density lipoprotein receptor (LDLR) is markedly reduced in SC4MOL or NSDHL-deficient cells. These effects were completely reversible with inactivation of LXR alpha, or via inactivation of an upstream enzyme, CYP51A1, which eliminated MAS. Inactivation of SC4MOL and NSDHL sterol pathway enzymes and upregulation of cholesterol efflux via ABCA1 depleted cholesterol from cellular membranes as evidenced by marked increase of nuclear form of SREBP2. Direct manipulation of SREBP, LXR or LDLR produced concordant synergistic effects with anti-EGFR drugs in carcinoma cell lines suggesting novel combinatorial strategies to treat EGFR-positive carcinomas. In in vivo experiments, SC4MOL or NSDHL-depleted A431 carcinoma xenografts showed exquisite sensitivity to cetuximab. This phenotype was associated with upregulation of LXR target ABCA1 and loss of LDLR. Conditional inactivation of a “floxed” Nsdhl allele via K14-Cre transgene triggered ABCA1 expression in the skin, arrested keratinocytes proliferation and caused lethality in newborn male pups. The anti-proliferative effect of NSDHL deficiency was tested in vivo against KRAS-driven tumors. Skin tumors induced by KRAS in transgenic LSL-KRasG12D mice are EGFR-dependent. Tamoxifen-inducible Tg(K5-CreERTam) mice were crossed with LSL-KRasG12D;NsdhlloxP/loxP and tumors induced by oral tamoxifen administration. Both, NsdhlloxP/Y (NSDHL-null) males carrying only the “floxed” allele and NsdhlloxP/+ heterozygous mosaic females formed skin tumors at similar rate. However, the growth of tumors in NSDHL-null males was completely abrogated. In sum, specific sterol metabolites play a fundamental role in regulating activity of oncogenic EGFR. Inhibition of SC4MOL or NSDHL is a highly effective strategy to counteract oncogenic signaling in carcinomas with activated EGFR-KRAS axis. Citation Format: Linara Gabitova, Andrey Gorin, Diana Restifo, Dong-Hua Yang, David Cunningham, Gail E. Herman, Igor A. Astsaturov. Meiosis activating sterols counteract KRas-driven epithelial carcinogenesis via an LXR-dependent mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2448. doi:10.1158/1538-7445.AM2014-2448