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The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG–asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1 . Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM.Significance:MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Figures S1 through S3

GSEA analysis

Supplementary Figure 1. Characterization of 23 PDAC cell lines.

Supplementary Figure 3. NVP-AUY922 toxicity in vivo and effect of gemcitabine treatment in ARID1A knock-out PDAC models in vitro and in vivo.

Comparison of IC50 values based on the gene mutation status

Supplementary Figure 2. Knocking out ARID1A does not impair viability in PDAC cell lines.

Cellular dedifferentiation is a key mechanism driving cancer progression. Acquisition of mesenchymal features has been associated with drug resistance, poor prognosis, and disease relapse in many tumor types. Therefore, successful targeting of tumors harboring these characteristics is a priority in oncology practice. The SWItch/Sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has also emerged as a critical player in tumor progression, leading to the identification of several SWI/SNF complex genes as potential disease biomarkers and targets of anticancer therapies. AT-rich interaction domain-containing protein 1A (ARID1A) is a component of SWI/SNF, and mutations in ARID1A represent one of the most frequent molecular alterations in human cancers. ARID1A mutations occur in approximately 10% of pancreatic ductal adenocarcinomas (PDAC), but whether these mutations confer a therapeutic opportunity remains unclear. Here, we demonstrate that loss of ARID1A promotes an epithelial–mesenchymal transition (EMT) phenotype and sensitizes PDAC cells to a clinical inhibitor of HSP90, NVP-AUY922, both in vitro and in vivo. Although loss of ARID1A alone did not significantly affect proliferative potential or rate of apoptosis, ARID1A-deficient cells were sensitized to HSP90 inhibition, potentially by promoting the degradation of intermediate filaments driving EMT, resulting in cell death. Our results describe a mechanistic link between ARID1A defects and a quasi-mesenchymal phenotype, suggesting that deleterious mutations in ARID1A associated with protein loss exhibit potential as a biomarker for patients with PDAC who may benefit by HSP90-targeting drugs treatment.Significance:This study identifies ARID1A loss as a promising biomarker for the identification of PDAC tumors that are potentially responsive to treatment with proteotoxic agents.

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K–AKT–MTOR pathway and by the activation of AXL, PDGFRa, and HER1–2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. Cancer Res; 75(6); 1091–101. ©2015 AACR.

supplementary Figures S1-S10. S1 growth and regression of iKRAS tumors. S2 culture comparison. S3 immunohistochemistry of p-erk and p-s6 S4 Phospho rtk array S5 Validation of anti AXL antibodies S6 validationn of the AXL inhibitor in vitro S7 validationn of the AXL inhibitor in vivo S8 efficacy of targeting AXL S9 RTK array of iKRAS cells treated with multiple RTK inhibitors S10 P-ERK levels

supplementary material and method

DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting ILF2, a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo an adaptive metabolic rewiring and rely on oxidative phosphorylation to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identified the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells’ ability to overcome ILF2 ASO−induced DNA damage, as being essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration. Our study revealed a novel vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation.STATEMENT OF SIGNIFICANCEMetabolic reprogramming is a mechanism through which cancer cells maintain survival and become resistant to DNA-damaging therapy. Here, we show that targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.

The lack of suitable experimental approaches to investigate and model functional heterogeneity in vivo has had a profound negative impact on our understanding of how heterogeneity affects the response to immunotherapy. To bridge this technological gap, we leverage a new platform that visualizes the spatial architecture of subclones and microenvironments. To study heterogeneous populations of cells and their clonal dynamics in vivo, we performed barcode lineage tracing with next-generation sequencing analysis. It allows a quantitative evaluation of the spatial distribution and temporal clonal dynamic in vivo with multiple pharmacological perturbations, such as immune checkpoint blockade (ICB). We found that treatment with a PD1 inhibitor, despite a limited effect on tumor volume, induced dramatic changes in tumor clonal architecture. Furthermore, we revealed that subclones from well-defined geographical domains in vivo share differential behavior upon immune checkpoint blockade (ICB). By visualizing 40 different markers with multiplexed staining technology, we further identified that the microenvironment also exhibits a high level of spatial heterogeneity. Therefore, we coupled spatial barcode sequencing with high-content imaging and revealed the differential microenvironment feature of sensitive and resistant clones. We will further explore the mechanisms of immune evasion at the subclonal level. Through isolation and deep characterization of clonal lineages endowed with a distinct ability to engage the immune response, we hope to identify new vulnerabilities to overcome resistance to ICB. Citation Format: Er-Yen Yen, I-Lin Ho, Chieh-Yuan Li, Charles Dyke, Shan Jiang, Francesca Citron, Sergio Attanasio, Rutvi Shah, Ko-Chien Chen, Giulio Draetta, Andrea Viale. Uncovering key microenvironmental features for immunotherapy response at the subclonal level in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5868.

Introduction: DNA replication is a tightly regulated, multi-step process involving licensing and replication factors that faithfully orchestrate a single duplication of the entire genome exclusively during S phase of the cell cycle. Although the mechanisms that prevent uncoordinated DNA replication are well characterized, it remains unclear how cells counteract its potentially harmful effects when it does occur, such as in cancer cells driven by aberrant oncogenic signaling and loss of cell cycle checkpoints. Here, we identify DPY30, an understudied component of all COMPASS complexes, as a modulator of DNA replication. We found upregulated DPY30 expression in pancreatic ductal adenocarcinoma (PDAC) as well as a correlation between increasing DPY30 expression level and higher tumor grade and poor clinical outcomes. Experimental procedure: Human- and mouse-derived PDAC cells silenced or knockout for DPY30 expression were generated and characterized for their in vivo growth in recipient mice with increasing level of immune pressure. Chromatin immunoprecipitation followed by massive parallel sequencing was used to measure the epigenetic landscape in DPY30 or WDR5 knockdown cells. Unbiased proteomics and transcriptomics analyses were performed to elucidate molecular mechanisms. Results: The canonical function of COMPASS complexes is to regulate histone methylation. Unexpectedly, DPY30 loss did not affect histone methylation patterns compared to WDR5 loss, the core member of the COMPASS-like complex. This prompted unbiased proteomics analysis, which showed that DPY30 interacts with members of the DNA helicase, possibly stabilizing the DNA replication fork progression. We found that DPY30 overexpression, by preventing Geminin ubiquitylation, strongly stabilizes its expression, which in turn suppresses Cdt1 activity in late S-phase, buffering the risk of uncoordinated DNA replication in G2/M phase. Consistently, DPY30 loss resulted in DNA re-replication and chromosomal instability, as manifested by DNA fiber and metaphasic spread. In vivo, whole-exome sequencing analysis of DPY30-knockout tumors revealed that DPY30 loss markedly increased mutational burden in immunodeficient mice. In syngeneic models, DPY30 loss strongly inhibited tumor growth, prolonged survival, and increased T cell infiltration of tumors. Moreover, the immune system selectively cleared tumor cells with complex karyotypes, and we observed improved anti-tumor efficacy with DPY30 knockdown and anti-PD-1 treatment compared to either condition alone. Conclusions: In summary, we have identified a previously undescribed function for DPY30 to stabilize the replisome machinery and prevent excessive DNA replication. Our findings indicate that, in PDAC, DPY30 promotes genome stability, thus providing a rationale for targeting DPY30 or its effector proteins in combination with immune-checkpoint inhibitors. Citation Format: Francesca Citron, Luigi Perelli, Yanshuo Chu, Sanjana Srinivasan, I-Lin Ho, Er-Yen Yen, Rutvi Shah, Sergio Attanasio, Luis Castillo Montanez, Mauro Di Pilato, Shan Jiang, Wantong Yao, Sisi Gao, Angela Deem, Giannicola Genovese, Linghua Wang, Virginia Giuliani, Andrea Viale, Giulio F. Draetta. DPY30 loss leads to DNA re-replication and immunoediting in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 303.

Cellular dedifferentiation is a key mechanism driving cancer progression. Acquisition of mesenchymal features has been associated with drug resistance, poor prognosis, and disease relapse in many tumor types. Therefore, successful targeting of tumors harboring these characteristics is a priority in oncology practice. The SWItch/Sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has also emerged as a critical player in tumor progression, leading to the identification of several SWI/SNF complex genes as potential disease biomarkers and targets of anticancer therapies. AT-rich interaction domain-containing protein 1A (ARID1A) is a component of SWI/SNF, and mutations in ARID1A represent one of the most frequent molecular alterations in human cancers. ARID1A mutations occur in approximately 10% of pancreatic ductal adenocarcinomas (PDAC), but whether these mutations confer a therapeutic opportunity remains unclear. Here, we demonstrate that loss of ARID1A promotes an epithelial–mesenchymal transition (EMT) phenotype and sensitizes PDAC cells to a clinical inhibitor of HSP90, NVP-AUY922, both in vitro and in vivo. Although loss of ARID1A alone did not significantly affect proliferative potential or rate of apoptosis, ARID1A-deficient cells were sensitized to HSP90 inhibition, potentially by promoting the degradation of intermediate filaments driving EMT, resulting in cell death. Our results describe a mechanistic link between ARID1A defects and a quasi-mesenchymal phenotype, suggesting that deleterious mutations in ARID1A associated with protein loss exhibit potential as a biomarker for patients with PDAC who may benefit by HSP90-targeting drugs treatment. Significance: This study identifies ARID1A loss as a promising biomarker for the identification of PDAC tumors that are potentially responsive to treatment with proteotoxic agents.

The orbital siphon is a novel concept for propellantless payload transfer from the surface of a rotating body to orbit. In the context of asteroid mining, the orbital siphon represents an efficient...

Single-cell RNA sequencing studies have suggested that total mRNA content correlates with tumor phenotypes. Technical and analytical challenges, however, have so far impeded at-scale pan-cancer examination of total mRNA content. Here we present a method to quantify tumor-specific total mRNA expression (TmS) from bulk sequencing data, taking into account tumor transcript proportion, purity and ploidy, which are estimated through transcriptomic/genomic deconvolution. We estimate and validate TmS in 6,590 patient tumors across 15 cancer types, identifying significant inter-tumor variability. Across cancers, high TmS is associated with increased risk of disease progression and death. TmS is influenced by cancer-specific patterns of gene alteration and intra-tumor genetic heterogeneity as well as by pan-cancer trends in metabolic dysregulation. Taken together, our results indicate that measuring cell-type-specific total mRNA expression in tumor cells predicts tumor phenotypes and clinical outcomes. ispartof: NATURE BIOTECHNOLOGY vol:40 issue:11 pages:1624-+ ispartof: location:United States status: published

Defining tumor cell immune evasion Mouse models used to study cancer often lack a full immune system, allowing implantation of human tumors into the mice. By contrast, naturally evolving tumors must contend with a fully functional immune system and its destruction of some of the cells (see the Perspective by Ho and Wood). Two groups now report studies on mouse models with a fully intact immune system. Martin et al . started with preexisting murine tumor cell lines and examined their continued evolution in vivo, whereas Del Poggetto et al . examined the development of new pancreatic tumors in the context of inflammation, as is often seen in human patients. In each study, the authors found that the immune system exerted a selective pressure on cells that would give rise to tumors, promoting the survival of those that had lost expression of tumor suppressor genes or activated a specific oncogene. The findings suggest a major role for the immune system in driving tumor evolution across multiple types of cancer. —YN

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development. Statement of significance PDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors., Arginine methylation by PRMTs is dysregulated in cancer. Here, the authors use functional genomics screens and identify PRMT1 as a vulnerability in pancreatic ductal adenocarcinoma, and further show that PRMT1 regulates RNA metabolism and coordinates expression of genes in cell cycle progression, maintaining genomic stability and tumour growth.

Future space ventures will likely require exploitation of near-Earth asteroid resources. Moreover, it can be envisaged that asteroids may host habitats in their interiors. In fact, a cavern inside an asteroid would be a natural radiation shield against cosmic radiation and may also serve as a confined environment for storage of mined material such as water ice or other processed volatiles such as propellants. To this end, this paper proposes to leverage the asteroid rotational self-energy to remove material from the asteroid interiors and create a spherical cavern, by means of the orbital siphon concept. The siphon is a chain of tether-connected payload masses (the asteroid material), which exploits the rotation of the asteroid for the delivery of mass from the asteroid to escape. Under certain conditions the siphon can be initiated to ensure self-sustained flow of mass from the asteroid to escape. A net orbital siphon effect is generated by connecting new payloads at the bottom of the chain while releasing the upper payloads. Key parameters are discussed, such as the required siphon dimension and the maximum size of the internal cavity that can be excavated, as a function of the asteroid rotational period. Moreover, assuming elastic material behaviour, a closed-form expression for the stress tensor is found and a failure criterion is used to identify regions in the asteroid interiors subjected to the larger stresses. It is shown that the conditions for failure are relaxed as the radius of the internal void increases.

Cancers can vary greatly in their transcriptomes. In contrast to alterations in specific genes or pathways, differences in tumor cell total mRNA content have not been comprehensively assessed. Technical and analytical challenges have impeded examination of total mRNA expression at scale across cancers. To address this, we developed a model for quantifying tumor-specific total mRNA expression (TmS) from bulk sequencing data, which performs transcriptomic deconvolution while adjusting for mixed genomes. We used single-cell RNA sequencing data to demonstrate total mRNA expression as a feature of tumor phenotype. We estimated and validated TmS in 5,015 patients across 15 cancer types identifying significant inter-individual variability. At a pan-cancer level, high TmS is associated with increased risk of disease progression and death. Cancer type-specific patterns of genetic alterations, intra-tumor genetic heterogeneity, as well as pan-cancer trends in metabolic dysregulation and hypoxia contribute to TmS. Taken together, our results suggest that measuring cell-type specific total mRNA expression offers a broader perspective of tracking cancer transcriptomes, which has important biological and clinical implications.

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2–4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and—in the case of PDAC—the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface—where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth—is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities. In an inducible mouse model of pancreatic ductal adenocarcinoma, the signalling defect that underlies 90% of these tumours causes increased cell-surface expression of syndecan 1, leading to misregulation of macropinocytosis, and linking the defective signalling with nutrient-salvage pathways.

Introduction: Pre-existing tumor heterogeneity may allow tumors to escape treatment via selection and expansion of drug-resistant clones. Recent discovery of KRAS targeted therapies (e.g. SOS1i, KRASG12Ci, etc.) has dramatically changed the clinical outlook for patients with KRAS mutant tumors. Here, we generated and utilized pancreatic clonal replica tumors (CRTs) to identify drug-resistant clones of KRAS inhibitors (e.g. SOS1i and MEKi, KRASG12Di). Deep clonal characterization of KRAS inhibition revealed a novel epigenetic vulnerability which may inform on unbiased combination strategies to prolong responses for pancreatic cancer patients. Experimental procedures: Our CRT platform combines high-complexity in vivo lineage tracing with quantitative assessments of adaptive responses to therapeutics. In this study we generated a KRASG12D-driven CRT xenograft model of pancreatic ductal adenocarcinoma and treated the CRTs with MEKi, SOSi, and MEKi + SOSi. Upon comprehensive analysis of the clonal composition associated with each drug treatment, we identified, isolated, and validated a collection of clones with differential response to MEKi + SOS1i or KRASG12Di. Deep molecular characterization of sensitive and resistance clones identified novel combination strategies to overcome resistance to KRAS inhibitors. Results: We found that KRAS inhibition greatly impacted CRT clonal composition and tumor heterogeneity. Well-controlled reconstitution experiments with CRT-informed treatment-naïve clones confirmed the pre-existing nature of the differential clonal response to KRAS inhibitors in pancreatic cancer. Transcriptomic profiling of clonal diversity further revealed a BRD3-driven molecular plasticity in resistant clones that may drive resistance to MEKi + SOS1i or KRASG12Di. Pharmacological combination of MEKi (trametinib) + SOS1i (BI-3406) or KRASG12Di (MRTX1133) with BETi (BI 894999 and dBET6) exhibited combination benefits in preclinical pancreatic cancer models. Conclusions: Our study suggests that pre-existing heterogeneous subclones with epigenetic plasticity contribute to escaping direct KRAS inhibition in pancreatic cancer and provides a new avenue to overcome such resistance by combining KRAS inhibitors with BET inhibitors. Citation Format: Hengyu Lyu, Rosalba Minelli, Charles E. Deckard, Sahil Seth, Justin Huang, Hong Jiang, Michael D. Peoples, Meggie Lam, Christopher A. Bristow, Daniel Gerlach, Ulrike Tontsch-Grunt, Chieh-Yuan Li, I-Lin Ho, Christopher P. Vellano, Marco H. Hofmann, Timothy P. Heffernan, Joseph R. Marszalek, Andrea Viale, Alessandro Carugo. Pancreatic clonal replica tumors display functional heterogeneity in response to KRAS pharmacological inhibition and reveal unique epigenetic vulnerabilities to overcome resistance [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 LB079.

The association between tumors and inflammation is a long-established clinical observation. Although many studies demonstrated that the inflammatory microenvironment can promote tumor growth through the activation of survival and proliferation programs in cancer cells, the reason why inflammation, an evolutionarily conserved response to damage aimed at reestablishing tissue integrity upon injury, might be integral to tumorigenesis still remains unknown. Pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by poor prognosis, represents a distinctive example of cooperation between inflammation and activated oncogenes. Frequently developed in a context of chronic pancreatitis, PDAC is always associated with an inflammatory microenvironment. As supported by a substantial body of evidence across a multitude of experimental models, when occurring in the context of pancreatitis, mutations of KRAS, the universal oncogenic driver of pancreatic cancer, lead to accelerated tumor development inducing the appearance of neoplastic precursor lesions, such as acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN), which can evolve into invasive tumors. Interestingly, preneoplastic pancreatic alterations, specifically ADM, have been previously identified in acute and chronic pancreatitis in the absence of oncogene activation. Investigating the effects of inflammation on normal pancreatic epithelial cells, we discovered that long after complete resolution a transient inflammatory event primes cells to cooperate with oncogenic KRAS. Indeed, upon recovery from a single acute inflammation, epithelial cells display an enduring adaptive response associated with sustained epigenetic and transcriptional reprogramming. Such adaptation facilitates the prompt reactivation of acinar-to-ductal metaplasia upon subsequent inflammatory events, representing a physiological mechanism for limiting tissue damage via rapid decrease of zymogen production. Because metaplastic lesions are mediated by the activation of MAPK signaling, we demonstrated that activating mutations of KRAS, maintaining an irreversible ADM, are protective against tissue damage in a contest of pancreatitis. Uncovering a new physiologic role of somatic oncogenic mutations in preserving tissue integrity during repeated damages, we propose that KRAS mutations represent a nearly universal event in pancreatic cancer because beneficial and under strong positive selection in the context of recurrent pancreatitis. Citation Format: I-Lin Ho, Edoardo Del Poggetto, Chiara Balestrieri, Er-Yen Yen, Francesca Citron, Rutvi Shah, Shaojun Zhang, Shan Jiang, Wantong Yao, Haoqiang Ying, Giannicola Genovese, Timothy Heffernan, Anirban Maitra, Linghua Wang, Timothy Wang, Giulio Draetta, Alessandro Carugo, Gioacchino Natoli, Andrea Viale. Epithelial memory of resolved inflammation cooperates with oncogenic KRAS to limit tissue damage while promoting pancreatic cancer [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 1327.

Radiotherapy (RT) plus the anti‐EGFR monoclonal antibody Cetuximab (CTX) is an effective combination therapy for a subset of head and neck squamous cell carcinoma (HNSCC) patients. However, predictive markers of efficacy are missing, resulting in many patients treated with disappointing results and unnecessary toxicities. Here, we report that activation of EGFR upregulates miR‐9 expression, which sustains the aggressiveness of HNSCC cells and protects from RT‐induced cell death. Mechanistically, by targeting KLF5, miR‐9 regulates the expression of the transcription factor Sp1 that, in turn, stimulates tumor growth and confers resistance to RT+CTX in vitro and in vivo. Intriguingly, high miR‐9 levels have no effect on the sensitivity of HNSCC cells to cisplatin. In primary HNSCC, miR‐9 expression correlated with Sp1 mRNA levels and high miR‐9 expression predicted poor prognosis in patients treated with RT+CTX. Overall, we have discovered a new signaling axis linking EGFR activation to Sp1 expression that dictates the response to combination treatments in HNSCC. We propose that miR‐9 may represent a valuable biomarker to select which HNSCC patients might benefit from RT+CTX therapy., The combination therapy of Radiotherapy + Cetuximab (RT + CTX) is currently used for the treatment of HNSCC. Its lower toxicity compared to chemotherapy makes it the primary choice for fragile patients. This study identifies miR‐9 as a biomarker of RT + CTX responsiveness and explains why miR‐9 may be especially relevant in TP53 mutated HNSCC.

Dedifferentiation and acquisition of chromosomal instability in renal cell carcinoma portends dismal prognosis and aggressive clinical behavior. However, the absence of reliable experimental models dramatically impacts the understanding of mechanisms underlying malignant progression. Here we established an in vivo genetic platform to rapidly generate somatic mosaic genetically engineerd immune-competent mouse models of renal tumors, recapitulating the genomic and phenotypic features of these malignancies. Leveraging somatic chromosomal engineering, we demonstrated that ablation of the murine locus syntenic to human 9p21 drives the rapid expansion of aggressive mesenchymal clones with prominent metastatic behavior, characterized by early emergence of chromosomal instability, whole-genome duplication, and conserved patterns of aneuploidy. This model of punctuated equilibrium provides a remarkable example of cross-species convergent evolution.SignificanceTo better understand the role of 9p21 in malignant progression, we generated a somatic mosaic GEMM of renal cancer, capturing the histological, genomic and evolutionary features of human disease. With this technology we demonstrated a critica role of 9p21 loss in metastatic evolution of RCC and provide a unique tool for testing new therapeutic treatments.

Cancers can vary greatly in their transcriptomes. In contrast to alterations in specific genes or pathways, the significance of differences in tumor cell total mRNA content is poorly understood. Studies using single-cell sequencing or model systems have suggested a role for total mRNA content in regulating cellular phenotypes. However, analytical challenges related to technical artifacts and cellular admixture have impeded examination of total mRNA expression at scale across cancers. To address this, we evaluated total mRNA expression using single cell sequencing, and developed a computational method for quantifying tumor-specific total mRNA expression (TmS) from bulk sequencing data. We systematically estimated TmS in 5,181 patients across 15 cancer types and observed close correlations with clinicopathologic characteristics and molecular features, where high TmS generally accompanies high-risk disease. At a pan-cancer level, high TmS is associated with increased risk of disease progression and death. Moreover, TmS captures tumor type-specific effects of somatic mutations, chromosomal instability, and hypoxia, as well as aspects of intratumor heterogeneity. Taken together, our results suggest that measuring total mRNA expression offers a broader perspective of tracking cancer transcriptomes, which has important clinical and biological implications.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. Significance: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer. This article is highlighted in the In This Issue feature, p. 2659

SUMMARYGlioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain.In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

Cancers are highly complex ecosystems composed of molecularly distinct sub-populations of tumor cells, each exhibiting a unique spectrum of genetic features and phenotypes, and embedded within a complex organ context. To substantially improve clinical outcomes, there is a need to comprehensively define inter- and intra-tumor phenotypic diversity, as well as to understand the genetic dependencies that underlie discrete molecular subpopulations. To this end, we integrated CRISPR-based co-dependency annotations with a tissue-specific co-expression network developed from patient-derived models to establish CoDEX, a framework to quantitatively associate gene-cluster patterns with genetic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Using CoDEX, we defined multiple prominent anticorrelated gene-cluster signatures and specific pathway dependencies, both across genetically distinct PDAC models and intratumorally at the single-cell level. Of these, one differential signature recapitulated the characteristics of classical and basal-like PDAC molecular subtypes on a continuous scale. Anchoring genetic dependencies identified through functional genomics within the gene-cluster signature defined fundamental vulnerabilities associated with transcriptomic signatures of PDAC subtypes. Subtype-associated dependencies were validated by feature-barcoded CRISPR knockout of prioritized basal-like-associated genetic vulnerabilities (SMAD4, ILK, and ZEB1) followed by scRNAseq in multiple PDAC models. Silencing of these genes resulted in a significant and directional clonal shift toward the classical-like signature of more indolent tumors. These results validate CoDEX as a novel, quantitative approach to identify specific genetic dependencies within defined molecular contexts that may guide clinical positioning of targeted therapeutics.

Background & Aims Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. Methods To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. Results Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. Conclusions Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

SummaryPancreatic ductal adenocarcinoma (PDA) is characterized by a heterogenous and densely fibrotic microenvironment. This limits functional vasculature and diffusion of nutrients through the tumor1,2. Accordingly, pancreatic cancer cells develop numerous metabolic adaptations to survive and proliferate in nutrient austere conditions3-7. Subtypes of PDA have been characterized by transcriptional and functional differences8-12, which have been reported to exist within the same tumor13-15. However, it remains unclear if this diversity extends to metabolic programming. Here, using a combination of metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses within neoplastic populations isolated from a single pancreatic tumor. Furthermore, these populations are poised for metabolic crosstalk, and in examining this, we find an unexpected role for asparagine in maintaining cell proliferation following mitochondrial inhibition. Functionally, when challenged by mitochondrial inhibition, asparagine supplementation increases intracellular levels of asparagine and aspartate, a rate limiting biosynthetic precursor16-18. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes pancreatic tumors to mitochondrial targeting with phenformin. Together, these data extend the concept of metabolic diversity to neoplastic populations within individual tumors, while illustrating a new method of intratumoral communication that supports tumor fitness19,20. Finally, the combination of asparaginase with mitochondrial inhibition could provide a powerful new strategy for this difficult to treat disease.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable disease characterized by poor survival, dense desmoplastic stroma and activating mutations in KRAS ( Citation Format: Sanjana Srinivasan, Johnathon Rose, Wantong Yao, Sahil Seth, Michael Peoples, Annette Machado, Chieh-Yuan Li, I Lin Ho, Jaewon J. Lee, Paola A. Guerrera, Eiru Kim, Mustafa Syed, Joseph Daniele, Angela K. Deem, Michael Kim, Christopher A. Bristow, Eugene Koay, Giannicola Genovese, Andrea Viale, Timothy P. Heffernan, Anirban Maitra, Traver Hart, Alessandro Carugo, Giulio Draetta. Diversity across the pancreatic ductal adenocarcinoma disease spectrum revealed by network-anchored functional genomics [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 2136.