Andrea Viale

Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (KrasG12D, herein KRas) in a p53LoxP/WT background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.

Yap1 activation enables bypass of oncogenic KRAS addiction in pancreatic cancer

Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

Tumors and Mitochondrial Respiration: A Neglected Connection

For decades, tumor cells have been considered defective in mitochondrial respiration due to their dominant glycolytic metabolism. However, a growing body of evidence is now challenging this assumption, and also implying that tumors are metabolically less homogeneous than previously supposed. A small subpopulation of slow-cycling cells endowed with tumorigenic potential and multidrug resistance has been isolated from different tumors. Deep metabolic characterization of these tumorigenic cells revealed their dependency on mitochondrial respiration versus glycolysis, suggesting the existence of a common metabolic program active in slow-cycling cells across different tumors. These findings change our understanding of tumor metabolism and also highlight new vulnerabilities that can be exploited to eradicate cancer cells responsible for tumor relapse.

Telomere Dysfunction Drives Aberrant Hematopoietic Differentiation and Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime ‘collateral lethality’ therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease.

Genetic Events That Limit the Efficacy of MEK and RTK Inhibitor Therapies in a Mouse Model of KRAS-Driven Pancreatic Cancer

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.

Synthetic vulnerabilities of mesenchymal subpopulations in pancreatic cancer

Malignant neoplasms evolve in response to changes in oncogenic signalling. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1–Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α–MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

Sugar? No Thank You, Just a Deep Breath of Oxygen for Cancer Stem Cells

Tumors are metabolically heterogeneous, and subpopulations of tumorigenic cells have been recently described to rely more on mitochondrial respiration than glycolysis for energy production. In this issue, Sancho et al. (2015) demonstrate that MYC is a master switch regulating metabolic programs in different subpopulations of pancreatic tumor cells.

Abstract A03: Perturbation of proteostasis is lethal in SMARCB1-deficient tumors

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. The SWI/SNF complex, specifically, is involved in a plethora of biologic functions including cell cycle regulation, terminal differentiation, and regulation of cell metabolism. The crucial chromatin-remodeling function of the SWI/SNF complex during organogenesis and tissue specification is further supported by clinical data showing that the biallelic inactivation of the core subunits SMARCB1 and SMARCA4 results in the emergence of extremely aggressive pediatric malignancies characterized by a dramatic impairment of cell cycle regulation and cellular differentiation programs, resulting in highly lethal diseases characterized by early onset, widespread metastatic dissemination, and resistance to chemotherapy. So far the lack of conditional genetic models of malignant rhabdoid tumors (MRTs) made difficult to investigate the molecular bases of malignant transformation as well as the existence of dependencies associated with SMARCB1 loss. In order to identify the functional vulnerabilities of SMARCB1-deficient cancers, we developed an embryonic mosaic model of malignant rhabdoid tumors (MRTs) that faithfully recapitulates the clinicopathologic features of human disease. By using this novel experimental system we discovered that, upon SMARCB1 ablation, embryonic epithelial progenitors undergo a profound anabolic reprogramming resulting in a global increase in protein biosynthesis and in the adaptive activation of UPR and ER stress response. As a consequence, murine and human SMARCB1-deficient malignancies display an exquisite sensitivity to agents inducing proteotoxic stress and to the pharmacologic and genetic perturbation of autophagy. Our findings, therefore, have immediate clinical implications, paving the way for drug repositioning trials investigating combinations of agents with already known safety profiles targeting simultaneously UPR and the authopagic catabolic machinery in a class of orphan diseases affecting children with limited therapeutic options. Note: This abstract was not presented at the conference. Citation Format: Giannicola Genovese, Alessandro Carugo, Rosalba Minelli, Frederick, Scott Robinson, Pavlos Msaouel, Tim Heffernan, Andrea Viale, Nizar Tannir, Giulio Draetta. Perturbation of proteostasis is lethal in SMARCB1-deficient tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A03.

Abstract 4971: IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production to support cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-010759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that AML, plus subsets of glioblastoma, neuroblastoma, lymphoma, melanoma, triple negative breast cancer (TNBC) and pancreatic cancer (PDAC) are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in several cancer types with IACS-010759 led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. Mechanistically, response to IACS-010759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, aspartate biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. Tumor growth inhibition and regression have been observed in molecularly defined subsets of TNBC and PDAC PDX xenograft models treated with IACS-010759, indicating that subsets of these indications are dependent on OXPHOS. Furthermore, treating TNBC or PDAC PDX models post-chemotherapy with IACS-010759 extends progression free survival, consistent with IACS-010759 targeting recently described metabolically adapted residual tumor cells. In orthotopic xenograft models of primary AML cells, daily oral treatment with 1-7.5 mg/kg IACS-010759 extended the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and a gene signature levels. Therefore, these readouts (OCR, aspartate and a nanostring geneset) have been validated for use as exploratory clinical biology of response endpoints. In parallel, completion of preclinical chemistry, manufacturing and control (CMC) as well as GLP safety and tolerability studies with IACS-010759 in multiple species have enabled the selection of a clinical entry dose. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, a Phase I clinical trial in relapsed, refractory AML was initiated in October 2016, with a parallel trial in solid tumors expected to initiate in early 2017. Initial results from the on-going AML trial will be disclosed. Citation Format: Jennifer Molina, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Jason Cross, Naval Daver, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Jing Han, Judy Hirst, Sha Huang, Yongying Jiang, Zhijun Kang, Marina Konopleva, Gang Liu, Helen Ma, Polina Matre, Timothy McAfoos, Funda Meric-Bernstam, Pietro Morlacchi, Florian Muller, Marina Protopopova, Melinda Smith, Sonal Sonal, Yuting Sun, Jay Theroff, Andrea Viale, Quanyun Xu, Carlo Toniatti, Giulio Draetta, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors [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 4971. doi:10.1158/1538-7445.AM2017-4971