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Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

Oxidative phosphorylation (OXPHOS) is an active metabolic pathway in many cancers. RNA from pretreatment biopsies from patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy demonstrated that the top canonical pathway associated with worse outcome was higher expression of OXPHOS signature. IACS-10759, a novel inhibitor of OXPHOS, stabilized growth in multiple TNBC patient-derived xenografts (PDX). On gene expression profiling, all of the sensitive models displayed a basal-like 1 TNBC subtype. Expression of mitochondrial genes was significantly higher in sensitive PDXs. An in vivo functional genomics screen to identify synthetic lethal targets in tumors treated with IACS-10759 found several potential targets, including CDK4. We validated the antitumor efficacy of the combination of palbociclib, a CDK4/6 inhibitor, and IACS-10759 in vitro and in vivo. In addition, the combination of IACS-10759 and multikinase inhibitor cabozantinib had improved antitumor efficacy. Taken together, our data suggest that OXPHOS is a metabolic vulnerability in TNBC that may be leveraged with novel therapeutics in combination regimens. Significance: These findings suggest that triple-negative breast cancer is highly reliant on OXPHOS and that inhibiting OXPHOS may be a novel approach to enhance efficacy of several targeted therapies.

The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p Disclosures Jabbour: Pfizer: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding. Teachey:Sobi: Consultancy; Amgen: Consultancy; Janssen: Consultancy; La Roche: Consultancy. Rezvani:Takeda: Other: Licensing agreement; GemoAb: Membership on an entity's Board of Directors or advisory committees; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Virogen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Other: Educational grant; Affimed: Other: Educational grant; Formula Pharma: Membership on an entity's Board of Directors or advisory committees. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding. Lorenzi:Precision Pathways: Consultancy. Konopleva:Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Sanofi: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Rafael Pharmaceutical: Research Funding; Eli Lilly: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Agios: Research Funding; AstraZeneca: Research Funding; Ablynx: Research Funding; Forty-Seven: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Ascentage: Research Funding.

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (��-CTLA-4 or ��-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (>4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten���/���). Short-term treatment with ��-PD-1 and ��-CTLA-4 monotherapies were relatively ineffective, while treatment with ��-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined ��-OX40/��-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/��-CTLA-4/��-PD-1 or DT/��-OX40/��-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies.

Triple-negative breast cancer (TNBC) accounts for 15–20% of breast cancer cases in the United States, lacks targeted therapeutic options, and is associated with a 40–80% risk of recurrence. Thus, identifying actionable targets in treatment-naïve and chemoresistant TNBC is a critical unmet medical need. To address this need, we performed high-throughput drug viability screens on human tumor cells isolated from 16 patient-derived xenograft models of treatment-naïve primary TNBC. The models span a range of TNBC subtypes and exhibit a diverse set of putative driver mutations, thus providing a unique patient-derived, molecularly annotated pharmacologic resource that is reflective of TNBC. We identified therapeutically actionable targets including kinesin spindle protein (KSP). The KSP inhibitor targets the mitotic spindle through mechanisms independent of microtubule stability and showed efficacy in models that were resistant to microtubule inhibitors used as part of the current standard of care for TNBC. We also observed subtype selectivity of Prima-1Met, which showed higher levels of efficacy in the mesenchymal subtype. Coupling pharmacologic data with genomic and transcriptomic information, we showed that Prima-1Met activity was independent of its canonical target, mutant p53, and was better associated with glutathione metabolism, providing an alternate molecularly defined biomarker for this drug.

Treatment with molecularly-targeted therapy has revolutionized cancer care, including BRAF/MEK-targeted melanoma therapy. However responses are heterogenous and frequently not long-lasting. Novel strategies to target resistance are needed. We studied a cohort of patients with resectable metastatic melanoma treated with neoadjuvant BRAF/MEK-targeted therapy (n=52) and noted a strong sexual dimorphism in response to treatment, with female patients demonstrating significantly higher rates of a major pathologic response (MPR) (p=0.0001). RNA sequencing of tumors demonstrated enrichment of androgen-related genes in those failing to achieve MPR. Pre-clinical studies validated these findings, with significantly increased tumor growth in male vs female mice treated with BRAF/MEK inhibitors (BRAF/MEKi) (p=0.0005). Androgen receptor (AR) expression was upregulated in tumors of BRAF/MEKi-treated mice, and modulation of AR signaling via AR-blockade or castration was associated with significantly slower tumor growth (p=0.0001 and p=0.00004, respectively). Together, these results have important implications in the context of treatment with BRAF/MEKi-targeted therapy.

The SOS1i::KRAS inhibitor BI 1701963 is the first direct RAS signaling modifier to enter phase I clinical trials both as a monotherapy as well as in combination. SOS1::KRAS inhibitors exhibit activity against a broad spectrum of KRAS alleles, including the major G12D/V/C and G13D oncoproteins, while sparing the interaction of KRAS with SOS2. Here, we present pre-clinical data showing enhanced pathway modulation and synergistic anti-tumor effects following vertical pathway inhibition of BI 1701963 in combination with mitogen-activated protein kinase inhibitors (MEKi; trametinib and BI 3011441) or KRAS G12C inhibitors (MRTX849 and BI 1823911). Furthermore, SOS1::KRAS inhibitor treatment sensitizes tumor cells to increased DNA damage in combination with irinotecan. Our results highlight the suitability of SOS1::KRAS inhibitors as a backbone in combination therapies targeting KRAS-dependent tumors. Pre-clinical combination data supported the start of multiple phase I trials investigating the safety, tolerability, recommended dose and preliminary efficacy of BI 1701963 alone and in combination with other anti-cancer agents. Combination trials of BI 1701963 with MEKi include cohorts of patients with KRAS mutant solid tumors, such as non-small cell lung cancer (NSCLC), colorectal cancer (CRC), cholangiocarcinoma and pancreatic adenocarcinoma. Trials exploring the combination of BI 1701963 with irreversible KRAS G12C inhibitors (MRTX849 and BI 1823911) will include cohorts of patients with KRAS G12C mutant NSCLC and CRC. In a further study, the combination of BI 1701963 with irinotecan is being evaluated in patients with KRAS mutant CRC. Primary endpoints include dose-limiting toxicities, treatment-emergent or -related adverse events. Secondary endpoints include pharmacokinetic and pharmacodynamic properties of combination regimens and preliminary efficacy. Citation Format: Marco H. Hofmann, Hengyu Lu, Ulrich Duenzinger, Daniel Gerlach, Francesca Trapani, Annette A. Machado, Joseph R. Daniele, Irene Waizenegger, Michael Gmachl, Dorothea Rudolph, Christopher P. Vellano, Marcelo Marotti, Vitomir Vucenovic, Timothy P. Heffernan, Joseph R. Marszalek, Mark P. Petronczki, Norbert Kraut. Trial in Process: Phase 1 studies of BI 1701963, a SOS1::KRAS Inhibitor, in combination with MEK inhibitors, irreversible KRASG12C inhibitors or irinotecan. [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 CT210.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal adult cancers with an average 5-year survival rate of less than 10% due in part to the limited number of effective therapies. Activation of TRAILR2 (Tumor necrosis factor (TNF)-Related Apoptosis-Inducing Ligand Receptor 2) has emerged as an important therapeutic concept in cancer treatment. Traditional TRAILR2 agonists have had limited clinical success due to lack of efficacy or, importantly, severe hepatotoxicity. Here we present anti-tumor activity in preclinical PDAC models for BI 905711, a first-in-class tetravalent bispecific antibody specifically designed to overcome the disadvantages of previous strategies targeting TRAILR2. BI 905711 serves as a uniquely specific, and liver-sparing therapeutic by targeting tumors that co-express TRAILR2 and another cell surface protein CDH17, which has ~40% prevalence in PDAC and is not expressed in normal liver. Working from a large cohort of molecularly characterized PDAC PDX models, we provide the first preclinical evidence of BI 905711 exhibiting robust anti-tumor activity in difficult to treat PDAC PDX models. Anti-tumor efficacy in responding models correlated with strong induction of Caspases 3/7 and 8 activation in tumors 24 hours after a single dose of BI 905711, and was associated with the presence and expression levels of TRAILR2 and CDH17 proteins. Evaluation of models with differential TRAILR2 and CDH17 expression profiles helped define the expression threshold for each target that is associated with response, upon which clinical assay development is in process for future patient stratification. Additionally, response was also associated with PDAC molecular subtypes utilizing a novel proprietary gene co-expression network developed from a curated cohort of PDAC PDX tumors. Responders to BI 905711 were identified primarily within the classical and quasi-basal/hybrid subtypes when TRAILR2 was adequately co-expressed. This correlates with an enrichment pattern of CDH17 gene expression that is mostly within the classical gene cluster and strongly anti-correlated with basal-like cluster enrichment. Robust preclinical anti-tumor activity of BI 905711 in TRAILR2 and CDH17-expressing PDAC PDX models, along with this antibody's potential for a favorable safety profile, has justified the enrollment of pancreatic cancer patients in the ongoing BI 905711 FIH Phase I clinical trial (NCT04137289). Citation Format: Jing Han, Annette A. Machado, Mikhila Mahendra, Joseph R. Daniele, Christopher A. Bristow, Justin Kwang-Lay Huang, Alessandro Carugo, Robert A. Mullinax, Benjamin J. Bivona, Ningping Feng, Poojabahen Gandhi, Norbert Schweifer, Paolo Maria Chetta, Juan Manuel Garcia-Martinez, Frank Hilberg, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek. BI 905711 selectively induces apoptosis and anti-tumor response in TRAILR2/CDH17- expressing pancreatic cancer 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 985.

Despite recent approval of hypomethylating agent/venetoclax (HMA+VEN) therapy for older patients (pt) with AML unfit for induction chemotherapy, their outcomes remain suboptimal. Such pts have a median overall survival of only 14 months and only approximately 35% of pts enjoying long-term survival (DiNardo, EHA 2020). Approximately 20-40% of newly diagnosed older pts with AML do not respond to the HMA+VEN regimens, with higher rates of refractory disease as well as early relapse in high-risk pts. Metabolic reprogramming and dependence on mitochondrial oxidative phosphorylation (OxPhos) is a core feature of AML leukemia stem cells (LSC). Recent reports have shown that upregulation of OxPhos confers intrinsic and acquired resistance to VEN in AML, multiple myeloma and lymphoid malignancies, which can be reversed by disrupting OxPhos; mutated TP53 has been shown to confer intrinsic resistance to venetoclax through increased OxPhos (Nechiporuk T, et al. Cancer Discovery 2019). We hypothesized that combined blockade of mitochondrial fitness by OxPhos inhibitors and of BCL-2 with VEN/azacitidine (AZA) will perform synergistically in pre-clinical AML models. To test this hypothesis, we utilized a novel complex I inhibitor IACS-010759 that effectively inhibits cell respiration and leukemia progression in the in vitro and in vivo AML pre-clinical models (Molina, Nat Med 2018). Priming of MOLM-13 cells with 20nM VEN for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while single agents reduced OCR by 70%) of viable cell numbers in several AML cell lines tested (OCI-AML2, MV-4-11, and MOLM-13) (Figure 1). By co-immunoprecipitation VEN disrupted interaction of BCL-2 with the mitochondrial protein VDAC, known to regulate ADP/ATP exchange during electron transport across mitochondria membrane; this resulted in dramatic reduction of the intracellular ATP and CTP levels measured by MS-based metabolomics. Further, VEN increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of VEN and IACS-010759, possibly because of RNA degradation. In primary AML samples (n=3) and AML PDX cells (n=4) cultured ex vivo, combined VEN and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. We next tested the efficacy of the "triple" combination of VEN, AZA and IACS-010759 in the in vivo AML PDX models. We injected AML PDX cells 3871344 (with no mutations identified by targeted sequencing) and 4404778 (harboring IDH1, NPM1, FLT3-ITD mutations) into NRG mice and upon engraftment, randomized mice into 4 groups to receive 2 cycles of treatment with 3 weeks interruption between cycles: vehicle, VEN (50mg/kg daily, 5 days on/2 days off, day 1-21) with AZA (2.5 mg/kg daily, day 1-7), IACS-010759 (1mg/kg ?daily, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. In the less aggressive model 3871344, all therapies reduced circulating leukemia burden, and the triple treatment achieved best efficacy, with average circulating tumor burden at 10 weeks after cycle 2 of 90.8%, 38.7%, 68.8% and 7.4% in vehicle, VEN+AZA, IACS-010759, and triple-therapy cohorts, respectively. In the aggressive model 4404778, the treatments were less effective, but the combination offered highest activity, with average circulating tumor burden of 71.0%, 52.3%, 88.7% and 39.9% in vehicle, IACS-010759, VEN+AZA and triple-therapy cohorts, respectively (Figure 1). Analysis of survival and additional PDX models are ongoing and will be reported. In summary, our study demonstrates that BCL-2 modulates mitochondrial respiration and mitochondrial ATP generation in addition to its established anti-apoptotic role. VEN disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitro and in vivo. Disclosures Konopleva: Ascentage: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; AstraZeneca: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding.

Eradicating triple negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard front-line chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Taken together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes as well as maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Finally, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase I clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by non-selective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.

PARP inhibitors have been proven clinically efficacious in platinum-responsive ovarian cancer regardless of BRCA1/2 status and in breast cancers with germline BRCA1/2 mutation. However, resistance to PARP inhibitors may preexist or evolve during treatment in many cancer types and may be overcome by combining PARP inhibitors with other therapies, such as immune checkpoint inhibitors, which confer durable responses and are rapidly becoming the standard of care for multiple tumor types. This study investigated the therapeutic potential of combining niraparib, a highly selective PARP1/2 inhibitor, with anti-PD-1 immune checkpoint inhibitors in preclinical tumor models. Our results indicate that niraparib treatment increases the activity of the type I (alpha) and type II (gamma) interferon pathways and enhances the infiltration of CD8+ cells and CD4+ cells in tumors. When coadministered in immunocompetent models, the combination of niraparib and anti-PD-1 demonstrated synergistic antitumor activities in both BRCA-proficient and BRCA-deficient tumors. Interestingly, mice with tumors cured by niraparib monotherapy completely rejected tumor growth upon rechallenge with the same tumor cell line, suggesting the potential establishment of immune memory in animals treated with niraparib monotherapy. Taken together, our findings uncovered immunomodulatory effects of niraparib that may sensitize tumors to immune checkpoint blockade therapies.

KRAS is the most frequently mutated oncogene with high prevalence of alterations in pancreatic, colorectal, and non-small cell lung tumors. The alleviation of negative feedback control of KRAS activity upon downstream inhibition has limited the clinical efficacy of MAPK pathway drugs in the KRAS mutant context. The guanine nucleotide exchange factor (GEF) SOS1 is a mediator of this negative feedback control, and the protein is a key activator of KRAS itself. BI 1701963 is the first SOS1::KRAS inhibitor to advance to clinical trials. Both BI 1701963 as well as the previously reported probe compound BI-3406 are orally bioavailable small molecules, designed to bind to the catalytic domain of SOS1, thereby preventing the interaction with KRAS-GDP. In KRAS-dependent cancers, both SOS1::KRAS inhibitors potently reduce the formation of GTP-loaded KRAS, and inhibit MAPK pathway signaling. Both SOS1::KRAS inhibitors exhibit activity on a broad spectrum of KRAS alleles, including all major G12D/V/C and G13D oncoproteins, while sparing the interaction of KRAS with SOS2. This increases the targetable patient population beyond KRAS G12C mutation and suggests a favorable therapeutic window to enable rational combinations. We previously showed that combining the SOS1::KRAS inhibitor BI-3406 with the MEK inhibitor trametinib blocks the negative feedback relief induced by MAPK inhibition. Combining SOS1::KRAS inhibition with trametinib treatment enhances pathway blockade and elicits tumor regressions in vivo in xenograft models driven by mutant KRAS. In this work, we present combination data for the clinical candidate BI 1701963 with the MEK inhibitor trametinib and, in addition, with irinotecan, a key component of the widely used CRC standard-of-care chemotherapy treatment regimen. Both combinations show strong in vivo efficacy in tumor mouse models. The SOS1::KRAS/MEK inhibitor combination results in anti-tumor effects in a broad spectrum of KRAS-mutated CRC PDX models. In addition, extensive biomarker data on the combination of the clinical candidate BI 1701963 with trametinib reveals time- and dose-dependent modulation of transcriptional target genes in the MAPK pathway. Furthermore, we present the first data on the therapeutic effectiveness of combining SOS1::KRAS inhibitors with irinotecan, as well as its underlying mechanism. The SOS1::KRAS inhibitor BI 1701963 is currently in a Phase I clinical trial. This multi-center trial is currently recruiting patients with advanced KRAS-mutated cancers to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and preliminary efficacy of BI 1701963 alone and in combination with trametinib. Citation Format: Daniel Gerlach, Michael Gmachl, Juergen Ramharter, Jessica Teh, Szu-Chin Fu, Francesca Trapani, Dirk Kessler, Klaus Rumpel, Dana-Adriana Botesteanu, Peter Ettmayer, Heribert Arnhof, Thomas Gerstberger, Christiane Kofink, Tobias Wunberg, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Marco H. Hofmann. BI-3406 and BI 1701963: Potent and selective SOS1::KRAS inhibitors induce regressions in combination with MEK inhibitors or irinotecan [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 1091.

BackgroundDespite outstanding responses to anti-PD-1 agents in a subset of non-small cell lung cancer (NSCLC) patients, approximately 80% of patients fail to have prolonged favorable response. Recent studies show that tumor cell oxidative metabolism is a barrier to PD-1 immunotherapy and radiotherapy could overcome PD-1 resistance, so it is urgent to determine if combination treatment with radiotherapy and a novel oxidative phosphorylation (OXPHOS) inhibitor (IACS-010759) is an effective strategy against PD-1 resistance in NSCLC.MethodsThe antitumor effect of this combinational treatment was evaluated in vitro and in vivo. For in vivo experiments, we treated 129Sv/Ev mice with anti-PD1-sensitive and anti-PD1-resistant 344SQ NSCLC adenocarcinoma xenografts with oral IACS-010759 combined with radiotherapy (XRT). In vitro experiments included PCR, seahorse bioenergetic profiling, flow cytometry phenotyping, and clonogenic survival assay.ResultsIn the current study, we found that our PD-1-resistant model utilized OXPHOS to a significantly greater extent than the PD-1-sensitive model and XRT increased OXPHOS in vitro and in vivo. Thus, we explored the effect of the novel OXPHOS inhibitor IACS-010759 on PD-1-resistant NSCLC in an effort to overcome XRT-induced immunosuppression and maximize response to PD-1. Additionally, combined XRT and IACS-010759 promoted antitumor effects in the PD-1-resistant model, but not in the sensitive model. After elucidation of the most optimal dose/fractionation scheme of XRT with IACS-010759, the combinatorial therapy with this regimen did not increase the abscopal antitumor effect, although IACS-010549 did not decrease CD45+, CD4+, and CD8+ immune cells. Finally, triple therapy with IACS-010759, XRT, and anti-PD-1 promoted abscopal responses and prolonged survival time.ConclusionOXPHOS inhibition as part of a combinatorial regimen with XRT is a promising strategy to address PD-1-resistant NSCLC, and this combination is being tested clinically.

There is a critical need to improve our understanding of the pathogenesis of melanoma brain metastases (MBM). Thus, we performed RNA sequencing on 88 resected MBMs and 42 patient-matched extracranial metastases; tumors with sufficient tissue also underwent whole-exome sequencing, T-cell receptor sequencing, and IHC. MBMs demonstrated heterogeneity of immune infiltrates that correlated with prior radiation and post-craniotomy survival. Comparison with patient-matched extracranial metastases identified significant immunosuppression and enrichment of oxidative phosphorylation (OXPHOS) in MBMs. Gene-expression analysis of intracranial and subcutaneous xenografts, and a spontaneous MBM model, confirmed increased OXPHOS gene expression in MBMs, which was also detected by direct metabolite profiling and [U-13C]-glucose tracing in vivo. IACS-010759, an OXPHOS inhibitor currently in early-phase clinical trials, improved survival of mice bearing MAPK inhibitor–resistant intracranial melanoma xenografts and inhibited MBM formation in the spontaneous MBM model. The results provide new insights into the pathogenesis and therapeutic resistance of MBMs. Significance: Improving our understanding of the pathogenesis of MBMs will facilitate the rational development and prioritization of new therapeutic strategies. This study reports the most comprehensive molecular profiling of patient-matched MBMs and extracranial metastases to date. The data provide new insights into MBM biology and therapeutic resistance. See related commentary by Egelston and Margolin, p. 581. This article is highlighted in the In This Issue feature, p. 565

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Exosomes are extracellular vesicles produced by all cells with a remarkable ability to efficiently transfer genetic material, including exogenously loaded siRNA, to cancer cells. Here, we report on a bioreactor-based, large-scale production of clinical-grade exosomes employing good manufacturing practice (GMP) standards. A standard operating procedure was established to generate engineered exosomes with the ability to target oncogenic Kras (iExosomes). The clinical-grade GMP iExosomes were tested in multiple in vitro and in vivo studies to confirm suppression of oncogenic Kras and an increase in the survival of several mouse models with pancreatic cancer. We perform studies to determine the shelf life, biodistribution, toxicology profile, and efficacy in combination with chemotherapy to inform future clinical testing of GMP iExosomes. Collectively, this report illustrates the process and feasibility of generating clinical-grade exosomes for various therapies of human diseases.

KRAS is the most frequently mutated oncogene with high prevalence in pancreatic, colorectal, and non-small cell lung tumors. KRAS signaling is tightly regulated and various factors, including negative feedback pathways have limited the clinical efficacy of inhibitors of downstream MAPK signaling in the KRAS mutant context. Here we report the discovery of BI-3406 and demonstrate it is a highly potent and selective, orally bioavailable SOS1::KRAS inhibitor which binds to the catalytic domain of the guanine nucleotide exchange factor (GEF) SOS1 thereby preventing the interaction with KRAS-GDP. BI-3406 does not block the interaction of KRAS with SOS2 but elicits activity on a broad panel of KRAS oncogenic variants, including all major G12 and G13 oncoproteins. In KRAS-dependent cancers, BI-3406 potently reduces the formation of GTP-loaded KRAS, and inhibits MAPK pathway signaling. Down-modulation of this signaling cascade by BI-3406 in KRAS G12 or G13 mutant cells effectively limits cell proliferation. As a monotherapy, BI-3406 modulates signaling, as assessed by p-ERK and target genes, and displays marked anti-tumor efficacy in KRAS mutant xenografts. Due to BI-3406 blocking the negative feedback relief induced by MAPK inhibition, it has the potential to sensitize KRAS-dependent cancers to MEK inhibitor treatment. Combination with MEK inhibition leads to profound pathway blockade and tumor regressions in vivo. The combination of SOS1 and MEK inhibition is a potential therapy for the majority of KRAS-driven cancers including those fuelled by the most prevalent KRAS mutant oncoproteins. Furthermore, the pharmacological properties of BI-3406 and close analogues hold the promise of a significant treatment benefit in a broad patient population that is currently lacking precision medicine options. A Phase 1 clinical trial is in preparation for patients with advanced KRAS-mutated cancers to evaluate safety, tolerability, pharmacokinetic and pharmacodynamic properties, and preliminary efficacy of BI 1701963, a SOS1::KRAS inhibitor closely related to BI-3406. Citation Format: Marco H Hofmann, Michael Gmachl, Jürgen Ramharter, Fabio Savarese, Daniel Gerlach, Joseph R Marszalek, Michael P Sanderson, Francesca Trapani, Dirk Kessler, Klaus Rumpel, Dana-Adriana Botesteanu, Peter Ettmayer, Heribert Arnhof, Thomas Gerstberger, Christiane Kofink, Tobias Wunberg, Szu-Chin Fu, Jessica Teh, Christopher P. Vellano, Jonathan C. O’Connell, Rachel L Mendes, Juergen Moll, Timothy P. Heffernan, Mark Pearson, Darryl B McConnell, Norbert Kraut. Discovery of BI-3406: A potent and selective SOS1::KRAS inhibitor opens a new approach for treating KRAS-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL06-01. doi:10.1158/1535-7163.TARG-19-PL06-01

Human papilloma virus (HPV) is an oncogenic driver for a subset of head and neck squamous cell carcinomas (HNSCC), primarily from the oropharyngeal tissue subsite (OPSCC). These tumors are increasing in incidence and have recently surpassed cervical cancer as the most common HPV-driven malignancy in the United States. Fortunately, these tumors generally respond well to radiation-based therapy (XRT), and long-term (5 yr) survival is around 85%. However, the XRT treatment can generate significant morbidity, including problems with speech and swallowing. There is a clinical effort to reduce the treatment-related morbidity without compromising survival outcomes, through de-escalation treatment protocols. However, there is a subset of HPV+ OPSCC patients who do not respond to the current therapies and should not be given less intense treatment. This has generated the need to stratify patients based on their risk of recurrence or death, but currently no molecular biomarkers are available for risk assessment in OPSCC. By analyzing genomic data from The Cancer Genome Atlas (TCGA) we have identified a gene expression signature associated with expression of HPV genes. This signature identified 2 groups within the HPV+ tumors that demonstrate different levels of HPV function. One group seems to have reduced HPV function and present with intermediate phenotypes between HPV+ and HPV- tumors. Importantly, this signature is also highly prognostic in HPV+ OPSCC (p Citation Format: Frederico O. Gleber-Netto, Meng Gao, Xiayu Rao, Christopher P. Vellano, Joseph R. Marszalek, Jing Wang, Faye M. Johnson, Curtis R. Pickering. Variations in HPV function are associated with patient outcome and identify new candidate therapeutic approaches [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 4942.

3014 Background: A subset of tumors possess genetic or microenvironmental alterations that render cells dependent on mitochondria oxidative phosphorylation (OXPHOS) for survival. IACS, a potent oral selective inhibitor of mitochondrial complex I, showed robust responses in multiple preclinical tumor models, providing strong rationale for clinical testing. Methods: Pts with advanced cancers received IACS in increasing dose levels (DL) using 3+3 dose escalation. 7-day QD induction of IACS was followed by maintenance weekly (QW) or twice weekly (BIW) dosing. Phamacokinetics (PK), lactate and pH were assessed serially. Paired tumor biopsies were assessed for pharmacodynamic and predictive biomarkers. Results: 18 pts were treated; M/F 16/2; ECOG PS 0/1: 3/15. Mean age 49 (23-69) yrs. Tumors comprised advanced colorectal (n = 4), castration resistant prostate cancer (CRPC) (n = 3), pancreatic (n = 2), other cancers (n = 9). DL1: 2mg QD 7 days induction/0.5mg QW maintenance (n = 3); DL2: 2.5mg QD 7 days/1mg QW (n = 3); DL3: 3mg QD 7 days/3mg QW (n = 3); DL4: 2.5mg QD 7 days/2.5mg BIW (n = 4); DL5: 2mg QD 7 days/2mg BIW (n = 5). IACS was well tolerated with 12 (67%) pts reporting G1-2 IACS related toxicities, such as raised lactate (n = 10), nausea (n = 8), fatigue (n = 7), vomiting (n = 5), myalgia (n = 4) and peripheral neuropathy (n = 4). 1 pt in DL3 and 2 pts in DL4 had ≥G3 IACS related toxicities, such as nausea (n = 2), vomiting (n = 1), raised lactate (n = 1), dehydration (n = 1), visual changes (n = 1), and peripheral neuropathy (n = 1). Raised lactate was not associated with acidosis. DL5 is now being expanded to assess the maximum tolerated dose (MTD). PK showed good oral bioavailability, with long T1/2 and low intrapatient variability. Cmax = 14nM on Day 7 at the end of DL5 induction phase, confirming biologically active doses. 7 pts had best response of RECIST stable disease. A pt with heavily pretreated CRPC achieved RECIST partial response with resolution of CRPC related pain. Conclusions: IACS is well tolerated with preliminary evidence of antitumor activity. MTD expansions include CRPC, TNBC, pancreatic cancer and molecularly selected (ENO1 loss; SMARCA4 mutation) tumor cohorts. Clinical trial information: NCT03291938.

Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by limited therapeutic options and a high rate of treatment failure due to chemoresistance. T-ALL is largely driven by activating NOTCH1 mutations, where oncogenic NOTCH1 facilitates glutamine oxidation, induces metabolic stress, and facilitates reliance on oxidative phosphorylation (OXPHOS)1. In other malignancies, the shift toward OXPHOS-dependent high-energy status is associated with acquired chemoresistance. In this study, we found that the novel inhibitor of mitochondrial complex I (OXPHOSi) IACS-0107592 has preclinical activity in NOTCH1-mutated T-ALL; we also characterize the cellular and metabolic responses to OXPHOS inhibition and propose that an OXPHOSi be incorporated into standard-of-care therapy to improve outcomes in patients harboring NOTCH1-mutated T-ALL. Exposure to IACS-010759 (0-370 nM) in vitro drastically reduced T-ALL viability, with EC50 ranging from 0.1-10 nM for cell lines (n=7) and from 13-60 nM for patient-derived xenograft (PDX)-derived and primary T-ALL cells (n=10) (Fig.1). Oral administration of IACS-010759 (7.5 mg/kg/day) significantly reduced leukemia burden and extended overall survival (p In summary, our findings indicate that OXPHOSi, alone and particularly in combination with standard chemotherapy and GLS inhibition, constitutes a novel therapeutic modality that targets a unique metabolic vulnerability of NOTCH1-mutated T-ALL cells. References:Kishton RJ, Barnes CE, Nichols AG at al., AMPK Is Essential to Balance Glycolysis and Mitochondrial Metabolism to Control T-ALL Cell Stress and Survival, Cell Metabolism, 2016, 23(4):649-62Molina JR, Sun Y, Protopopova M et al., An inhibitor of oxidative phosphorylation exploits cancer vulnerability, Nat Med, 2018, 24: 1036-1046 Disclosures Lorenzi: NIH: Patents & Royalties; Erytech Pharma: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

Introduction The majority of secondary central nervous system (CNS) lymphoma cases are derived from histologically high-grade systemic lymphomas such as diffuse-large B cell lymphoma (DLBCL) and Burkitt's lymphoma. These CNS lymphomas are associated with poor prognosis and a low likelihood of long-term survival, especially rituximab-ineffective CD20- lymphoma. To investigate the biological features and potential therapeutic targets of CNS-involved CD20- lymphoma, we established the first known patient-derived xenograft (PDX) model using cerebrospinal fluid (CSF). Methods Six- to 8-week-old male NSG mice (Jackson Laboratory) were housed and monitored in our animal research facility. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center. A total of 0.08 million cells were isolated from 2 mL of CSF obtained from a CD20- CD79A- DLBCL patient with Bcl-2 and Myc rearrangements (double-hit). The isolated cells were directly injected into a human fetal bone chip implanted into NSG (NSG-hu) mice. After passage, the mice were administered several small molecules, and tumor burden was measured via tumor volume and human β2M levels over the course of treatment. Kaplan-Meier survival analysis was also performed. PDX tissue was collected for histological analysis, immunophenotyping, genotyping, and proteomics analysis. Results The PDX tumors possessed the same histological, genetic and immunophenotypical features as the primary patient tumor cells. The PDXs had Bcl-2 and Myc rearrangements and were positive for CD10, CD19, CD22, and CD45 and were negative for CD5, CD20, and CD79A. After tumor passage, the mice were treated with vehicle, 50 mg/kg ibrutinib (Bruton's tyrosine kinase (BTK) inhibitor) oral gavage daily, 50 mg/kg ABT199 (Bcl-2 inhibitor) oral gavage daily, 1 mg/kg carfilzomib (proteasome inhibitor) i.v. twice per week, or 7.5 mg/kg IACS-010759 (oxidative phosphorylation (OXPHOS) inhibitor) oral gavage daily. As shown in Figure 1, the PDX tumor was resistant to the BTK inhibitor ibrutinib and moderately responsive to the proteasome inhibitor carfilzomib. Although the Bcl-2 gene was rearranged, the PDX model only had a partial response to the Bcl-2 inhibitor ABT-199 (ABT-199 vs control, p = 0.0004). In contrast, inhibition of the OXPHOS pathway with IACS-010759 significantly inhibited 96% tumor growth (IACS-010759 vs control, p < 0.00001) and prolonged mouse survival by approximately 25 days compared with the vehicle control and the other drugs (IACS-010759 vs control, ABT-199, or ibrutinib, p < 0.0001). Moreover, proteomics analysis demonstrated that IACS-010759 not only inhibited OXPHOS but also upregulated the pro-apoptotic protein Bim. Conclusion To our knowledge, this is the first PDX model generated from the CSF of a CD20- double-hit DLBCL patient. With this PDX model, we determined that the OXPHOS inhibitor IACS-010759 is a promising therapeutic agent for this aggressive cancer. Disclosures Wang: Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Novartis: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria; Juno: Research Funding; MoreHealth: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

A major barrier to achieving durable remission and definitive cure in oncology patients is the emergence of tumor resistance, a common outcome of different disease types independent from the therapeutic approach undertaken. Patients with pancreatic ductal adenocarcinoma (PDAC) continue to have a poor prognosis despite concerted efforts to advance new drugs to the clinic. One reason for this, in PDAC and other tumors, is that tumors are constantly adapting and evolving in response to external perturbations. To better investigate tumor evolution in response to therapy we developed a new clonal tracking platform that enables the in vivo study of long term self-renewing compartments and the generation of cohorts of patient-derived xenografts in which tumors are virtually identical and maintained by the same clones (clonal replica tumors), representing a unique tool to address fundamental questions about clonal dynamics in response to pharmacological treatment. Using this novel approach we demonstrate that standard of care in pancreatic cancer, despite inducing tumor regression, has minimal effect on the clonal composition of tumors that eventually relapse. Transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation shows that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate mitochondrial respiration (OXPHOS). Combining a novel inhibitor of oxidative phosphorylation (IACS-010759), developed at the MD Anderson Institute for Applied Cancer Science and currently in phase I clinical trial in relapsed/refractory acute myelogenous leukemia and advanced solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation. Citation Format: Sara Loponte, Denise Corti, Sahil Seth, Edoardo Del Poggetto, I-Lin Ho, Chieh-Yuan Li, Shan Jiang, Joseph R. Marszalek, Maria Emilia Di Francesco, Giannicola Genovese, Giulio Draetta, Alessandro Carugo, Andrea Viale. Metabolic targeting of chemoresistance perturbs clonal complexity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4953.

Fifty percent of patients with localized triple negative breast cancer (TNBC) have substantial residual cancer burden following treatment with neoadjuvant chemotherapy (NACT), resulting in a 40-80% risk of recurrence which leads to distant metastases and death for most patients. Intra-tumor heterogeneity (ITH) is a pervasive feature of TNBCs but the relative contributions of heterogeneous tumor cell populations to chemoresistance are not understood. To investigate the clonal dynamics that accompany chemotherapy treatment, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation. To monitor the fates of PDX tumor cell lineages undergoing treatment with front-line NACT, we treated multiple treatment-naïve PDX models with Adriamycin combined with Cytoxan (AC). Some PDXs initially exhibited partial sensitivity followed by maintenance of residual tumors that were resistant to chemotherapy. Residual tumors re-grew to regain partial chemo-sensitivity. To conduct barcode-mediated clonal tracking, we introduced a pooled lentiviral barcode library (Cellecta; 50M unique barcodes) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Strikingly, residual tumors maintained the same clonal architecture as untreated tumors. In contrast, only 20% of residual tumor clones repopulated tumors after discontinuation of treatment. Whole-exome sequencing revealed conservation of mutant allele frequencies throughout treatment. Together, these studies demonstrate that re-growth of residual tumors is accompanied by a non-random selection of subclones, that residual tumors surviving AC maintain the same levels of ITH as untreated tumors, and that selection of genomic subclones did not confer the observed chemoresistance. Transcriptomic, proteomic, and histologic profiling revealed that residual tumors exist in a distinct state characterized by alterations in EMT, metabolic, and cell adhesion programs. This state was reverted as tumors re-grew after discontinuation of AC treatment, indicating that the residual state may be a unique therapeutic window. In silico prediction of drug sensitivity revealed candidate drivers of resistance in the residual tumor state, and pharmacologic targeting identified multiple existing therapies that significantly delayed the regrowth of residual tumors. These data suggest that sequential administration of AC followed by these targeted agents could prolong TNBC responses, which may delay time to recurrence for patients with this highly aggressive disease. Citation Format: Gloria Vittone Echeverria, Sahil Seth, Zhongqi Ge, Alessandro Carugo, Christopher Bristow, Prabjhot Mundi, Sabrina Jeter-Jones, Xiaomei Zhang, Xinhui Zhou, Aaron McCoy, Shirong Cai, Yizheng Tu, Yuting Sun, Joseph Marszalek, Andrea Califano, William F. Symmans, Stacy L. Moulder, Jeffery T. Chang, Timothy P. Heffernan, Helen Piwnica-Worms. High-resolution barcoding in patient-derived xenografts of triple-negative breast cancer reveals reversible chemoresistance conferred by non-mutational mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 212.

Over the past few years we and others reported that specific populations of tumor cells including AML, subsets of lymphoma, glioblastoma, triple negative breast cancer (TNBC), melanoma and pancreatic ductal adenocarcinoma (PDAC) are highly dependent upon oxidative phosphorylation (OXPHOS) to meet their energy and biomass needs. Inhibition of OXPHOS in the context of these dependent tumor populations represents therefore an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign we discovered IACS-010759, a potent, selective small molecule inhibitor of complex I of the mitochondria electron transport chain that possesses excellent pharmacokinetic (PK) and pharmacologic properties, making it suitable for clinical development. We advanced IACS-010759 through IND studies and have recently initiated Phase I studies in patients with relapsed/refractory acute myeloid leukemia (AML) and advanced solid tumors and lymphomas (NCT02882321 and NCT03291938). In this presentation we will describe the identification of a novel series of Complex I inhibitors and their optimization into the clinical candidate compound IACS-010759. Several challenges were successfully overcome, including the optimization of the pharmacokinetic profile and the identification of inhibitors with minimal activity shift across preclinical species, thus enabling a thorough evaluation of the efficacy and toxicology profile. Aspects of the extensive translational research conducted to elucidate the mechanism of action of IACS-010759 and to position it into the clinic will be discussed, including the compelling pharmacological response observed in multiple PDX models of primary AML, and PDX xenograft models of lymphoma, TNBC, glioblastoma, melanoma and PDAC. The observed response was associated with robust pharmacodynamic read-out as assessed by modulation of oxygen consumption rate (OCR), aspartate and specific transcriptional changes. The presentation will also cover the preclinical development activities which resulted in IACS-010759 advancing into on-going phase 1 evaluation in AML and solid tumors. Citation Format: Maria Emilia Di Francesco, Joseph R. Marszalek, Timothy McAfoos, Christopher L. Carroll, Zhijun Kang, Gang Liu, Jay P. Theroff, Jennifer P. Bardenhager, Madhavi L. Bandi, Jennifer R. Molina, Sonal Gera, Marina Protopopova, Yuting Sun, Mary K. Geck Do, Ningping Feng, Jason P. Gay, Florian Muller, Marina Konopleva, Funda Meric-Bernstam, Carlo Toniatti, Timothy P. Heffernan, Giulio F. Draetta, Philip Jones. Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1655.

Fifty percent of all triple negative breast cancer (TNBC) patients harbor significant residual tumor burden following treatment with standard neoadjuvant chemotherapy (NACT), resulting in poor prognosis. Recent studies in TNBC have revealed extensive intra-tumoral heterogeneity at the time of diagnosis and throughout disease progression, but the relative contributions of these heterogeneous populations of tumor cells to chemoresistance are not well understood. The primary tumor, dermal metastasis, and germline reference were obtained from a patient with untreated metastatic TNBC. Tumor cells were engrafted into the humanized mammary fat pads of NOD/SCID mice to establish PDX models of the primary (PIM001-P) and metastatic (PIM001-M) tumors. RNA sequencing and whole-exome sequencing (WES), performed on the patient's primary and metastatic tumors and the first- and third- passage PDX models revealed transcriptomic profiles and subclonal heterogeneity of the patient's tumors were recapitulated in the PDX models. Treatment of mice engrafted with PIM001-P tumors with NACT (Adriamycin plus cyclophosphamide, AC) resulted in partial response, the magnitude of which was diminished in mice bearing PIM001-M tumors. Tumor subclones were tracked during chemotherapy treatment in mice engrafted with PIM001-P tumors using lentiviral non-targeting DNA barcodes. Residual tumors maintained the clonal architecture of untreated tumors, and deep WES revealed stable maintenance of somatic mutant allele frequencies throughout treatment. Therefore, selection of pre-existing resistant clones did not lead to AC resistance in this model. Interestingly, only 25% of residual tumor clones contributed to primary relapse once treatment was halted, suggesting only a subpopulation of tumor cells was able to reconstitute the tumor. RNA sequencing and reverse phase protein array revealed that while vehicle-treated and regrown tumors were highly similar, residual tumors harbored a unique profile characterized by numerous significant alterations in RNA and protein levels. Together, these results suggest that residual tumors enter into a transient drug-resistant state that is reversible. Residual tumors were enriched for alterations in pathways such as metabolism, extracellular matrix remodeling, and cell-cell communication. Pharmacologic targeting of the residual tumor state with an inhibitor of mitochondrial oxidative phosphorylation led to significant inhibition of tumor regrowth following AC treatment. Additional vulnerabilities identified in residual tumors are being targeted therapeutically with the goal of eradicating residual tumor cells. Citation Format: Echeverria GV, Seth S, Ge Z, Sun Y, DiFrancesco E, Lau R, Marszalek J, Moulder S, Symmans F, Heffernan TP, Chang JT, Piwnica-Worms H. Characterizing and targeting chemoresistant subclones in patient-derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-03-02.

Acute myeloid leukemia (AML) is a highly aggressive disease with a high mortality rate that encompasses several genetically and clinically diverse hematological malignancies characterized by clonal expansion of transformed stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of 65 years. Therefore, novel, highly effective therapeutics are needed for this population. Targeting bioenergetic susceptibilities is an exciting area of oncology therapeutics that is potentially applicable in AML. Our group and others have shown that AML blasts depend significantly on mitochondrial oxidative phosphorylation to meet their energy and biomass production demands. Through an extensive medicinal chemistry campaign IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain with excellent PK and a suitable overall profile. In AML cell lines and primary AML blasts treated ex vivo, we observe a robust decrease in proliferation and a concomitant increase in apoptosis with EC50 values of less than 10 nM. Response to IACS-10759 in AML cells was associated with induction of a metabolic catastrophe that negatively impacted the cells' ability to sustain energy homeostasis, amino acid biosynthesis, and nucleotide production. In a primary AML patient derived xenograft model from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 (QDx5/week) treatment at 10 mg/kg extended the median survival by greater than 2-fold. Inhibition of OXPHOS by IACS-10759 was confirmed in AML cell lines and PDX models by a decrease in oxygen consumption and significant changes in gene and protein expression, non-essential amino acids and nucleotides. Due to the robust response in AML cell lines, primary AML samples ex vivo, and in vivo efficacy in primary AML PDX models, IACS-10759 has been advanced through IND enabling studies with first-in-human studies targeted for the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Maria Alimova, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Sonal, Jay Theroff, Quanyun Xu, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A15.

Acute myeloid leukemia (AML) is a highly aggressive disease that is made up of several genetically and clinically diverse hematological malignancies that are characterized by clonal expansion of malignant stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of 65 years. Therefore, novel, highly effective therapeutics are needed for this population. Growing evidence suggests that in AML, metabolism is altered and that the tumor cells become highly dependent of mitochondria oxidative phosphorylation (OXPHOS) for their survival. We developed IACS-10759 as a novel small molecule inhibitor of complex I that potently inhibits oxygen consumption, eliminates hypoxia, and strongly inhibits the proliferation of cells grown in galactose medium with EC50 values between 1-10 nM. When AML cell lines as well as primary AML cells from relapsed/refractory patients were treated ex vivo with IACS-10759, apoptosis was robustly induced with EC50 values also ranging between 1-10 nM. It is noteworthy, that while apoptosis was induced in primary AML cells, normal patient-derived bone marrow cells were not sensitive. In AML orthotopic xenografts, daily oral dosing with 15 mg/kg IACS-10759 extended median survival to 70 days from 18 days in control animals. Taken together, the robust response in AML cell lines, primary AML samples ex vivo, and efficacy in orthotopic xenografts, IACS-10759 has entered IND enabling studies with first-in-human studies targeted for third quarter of 2015. Citation Format: Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christophor Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Florian Muller, Timothy Lofton, Timothy McAfoos, Jay Theroff, Yuting Sun, Yuanqiang Wu, Melinda Smith, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. Relapsed/refractory AML responds robustly to IACS-10759, a novel OXPHOS inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4455. doi:10.1158/1538-7445.AM2015-4455

Inhibition of mitochondria complex I in tumors that are metabolically dependent on oxidative phosphorylation (OXPHOS) for their survival offers unique synthetic lethal opportunities. Examples of dependent contexts are AML and DLBCL, where OXPHOS is highly active and subpopulations of glioblastoma and neuroblastoma that possess genetic alterations which make them glycolysis deficient. In addition, several lines of evidence indicate that after treatment with chemo or targeted therapy, residual tumor cells become reliant on OXPHOS for their continued survival. In each of these cellular states, excessive dependence on OXPHOS renders tumor cells vulnerable to therapeutic targeting strategies that exploit this addiction. We have generated a series of novel, highly potent complex I inhibitors, which in vitro inhibit complex I with IC50 values < 10 nM. When tested in cultured cell systems (cell lines and spheroids) these compounds inhibit oxygen consumption, eliminate hypoxia, and strongly inhibit the proliferation cells grown in galactose medium with EC50 values between 1-10 nM. Lead compounds specifically induce apoptosis with EC50 values between 1-10 nM in OXPHOS dependent cancer models such as AML and DLBCL cell lines and in glycolysis deficient cancer cell lines. Of note, apoptosis is induced in primary AML cells but not in normal patient-derived CD34+ cells. These compounds are orally bioavailable with excellent pharmacokinetics properties in preclinical species making them appropriate tools for proof-of-concept studies in vivo. In agreement with data in cell culture, we have shown that daily oral treatment with as low as 5 -10 mg/kg of our OXPHOS inhibitors is well tolerated and induce strong regression of NB-1 (glycolysis-deficient cells) subcutaneous and intracranial xenografts. We have also demonstrated that sustained pharmacological inhibition of OXPHOS induce regression of DLBCL subcutaneous models and dramatically increase mice survival in an OCI-AML3 orthotopic xenograft model. In addition to synthetic lethality in monotherapy, we are exploring whether OXPHOS inhibition can overcome resistance to radiotherapy, chemotherapy and specific targeted therapies. Taken together, these data strongly support the notion that inhibiting OXPHOS in hypersensitive populations could be a novel, innovative therapeutic approach and justifies evaluation of OXPHOS inhibitors in a clinical setting. Citation Format: Joseph R. Marszalek, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ninping Feng, Barbara Czako, Jason Gay, Mary Geck Do, Jennifer Greer, Ryan M. Johnson, Marina Konopleva, Zhijun Kang, Gang Liu, Timothy Lofton, Timothy McAfoos, Marina Protopopova, Alessia Petrocchi, Florian Muller, Jay Theroff, Yuanqing Wu, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, Emilia Di Francesco. Identification of OXPHOS inhibitors which selectively kill tumors with specific metabolic vulnerabilities. [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 949. doi:10.1158/1538-7445.AM2014-949

Focal Adhesion Kinase (FAK) is a key regulator of cancer cell migration and invasion and overexpression of FAK and Src has been associated with resistance to current anticancer therapies. Thus, inhibition of FAK may represent a promising therapeutic approach to anti-cancer treatment. We investigated the anti-cancer activity of the FAK inhibitor (GSK2256098) in a panel of 26 human glioblastoma (GBM) cell lines. GBM is one of the most aggressive and lethal forms of cancer, characterized by exponential growth and diffuse invasiveness. Herein, we report that 8/26 GBM cell lines displayed sensitivity to FAK inhibition in both migration and Matrigel invasion assays. GSK2256098 had minimal effect on 2D cell proliferation. Integrated genomic and proteomic analyses revealed up-regulation of the PI3K and MAPK pathways in the FAK inhibitor-resistant GBM cell lines, suggesting possible drug combination strategies. To test this hypothesis, we assessed the effects of combining GSK2256098 with a PI3K inhibitor and a MEK inhibitor in the FAK inhibitor-resistant GBM cell lines. Our results suggest that multi-agent inhibition of FAK and MEK or PI3K may provide an attractive therapeutic strategy for GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3714. doi:1538-7445.AM2012-3714