Molecular Vulnerability Research Articles

HNF4A Defines Molecular Subtypes and Vulnerability to Transforming Growth Factor β-Pathway Targeted Therapies in Cancers of the Distal Esophagus

The transforming growth factor β (TGFβ) pathway exhibits pro-tumorigenic effects across multiple malignancies via its impact on tumor cells, microenvironment, and/or the immune system.1,2 Therefore, there is a growing impetus to target the TGFβ signaling axis across malignancies.1,2 Although early-phase/dose-defining trials with TGFβR1 (ALK5) inhibitor in glioma3 and TGFβ2 oligo therapy in anaplastic astrocytoma4 have shown improved outcomes, albeit in a small subset of patients, the molecular determinants that dictate response to TGFβ-pathway targeted therapies, however, remain elusive; underscoring a broader unmet need in precision oncology.

Abstract 272: Effective treatment of colorectal peritoneal metastases by exploiting a molecular subtype specific vulnerability

Abstract In colorectal cancer, peritoneal metastases (PMs) associate with severe morbidity and dismal prognosis. Given the incidence of this disease and the lack of adequate treatments currently available, PMs pose a large unmet clinical need. Although PMs can be accompanied by more widespread metastatic disease, it often occurs as the only sign of dissemination. This implies that the route of metastatic spread to the peritoneum differs from that to distant organs. PMs are thought to result from cancer cells that spill into the abdominal cavity, and are able to attach to the peritoneal lining and form tumor deposits. This cascade places specific demands on the cancer cells. Here, we report that colorectal cancers that present with PMs almost universally classify as consensus molecular subtype 4 (CMS4). This previously recognized disease entity is characterized by mesenchymal features, poor prognosis, and resistance to therapies currently used against peritoneal metastases, which explains their limited efficacy. By leveraging disease models that capture CMS4-specific features, including the ability to form PMs in vivo, we identified elesclomol as a highly effective agent. Elesclomol kills cancer cells in a copper-dependent fashion by targeting the oxidative phosphorylation machinery, which we found to be a specific vulnerability of CMS4 cancers. Elesclomol-Cu2+ was effective following only minutes of exposure to CMS4 cell lines and organoids, supporting its use in intra-abdominal treatment procedures. It is therefore a promising candidate for the local treatment of peritoneal metastases of colorectal cancer. Citation Format: Sanne Bootsma, Mark P. Dings, Leandro Ferreira Moreno, Kristiaan J. Lenos, Louis Vermeulen, Maarten F. Bijlsma. Effective treatment of colorectal peritoneal metastases by exploiting a molecular subtype specific vulnerability [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 272.

ATRT-23. SIRT2 cooperates with SMARCB1 to induce a differentiation block in ATRT

Atypical Teratoid Rhabdoid Tumor is a highly aggressive pediatric brain tumor with poor prognosis driven by loss of the chromatin remodeling factor SMARCB1 that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. We investigated the effect of SIRT2 inhibition in ATRT which was identified as a primary dependency in ATRT. SIRT2 inhibition with shRNA or Thiomyristoyl (TM) decreased ATRT cell growth, inhibited clonogenic potential and leaded to the cell cycle arrest. SIRT2 inhibition effectively suppresses pluripotency-associated genomic programs, significantly changed stem cell frequency, decreased tumor-sphere formation of ATRT cells and attenuated tumor cell self-renewal. In vivo SIRT2 inhibition decreased oncogenic markers and increased accumulation neuronal differentiation markers. Furthermore, SIRT2 induced apoptosis, decreased tumor growth and prolonged survival in orthotopic xenograft models. Single-cell RNA transcriptome analysis of xenoftaft tumors reveals elimination of tumor cells expressing stem cell genes and expansion of tumor cells expressing differentiated genes following TM treatment in ATRT. We demonstrated that SIRT2 inhibition is a molecular vulnerability in SMARCB1-deleted ATRT.

Induction of Fibroblast Growth Factor Receptor 4 by Helicobacter pylori via Signal Transducer and Activator of Transcription 3 With a Feedforward Activation Loop Involving SRC Signaling in Gastric Cancer

Helicobacter pylori (H pylori) infection is the main risk factor for gastric cancer. The role of fibroblast growth factor receptors (FGRFs) in H pylori-mediated gastric tumorigenesis remains largely unknown. This study investigated the molecular and mechanistic links between H pylori, inflammation, and FGFR4 in gastric cancer. Cell lines, human and mouse gastric tissue samples, and gastric organoids models were implemented. Infection with H pylori was performed using invitro and invivo models. Western blot, real-time quantitative reverse-transcription polymerase chain reaction, flow cytometry, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and luciferase reporter assays were used for molecular, mechanistic, and functional studies. Analysis of FGFR family members using The Cancer Genome Atlas data, followed by validation, indicated that FGFR4 messenger (m)RNA was the most significantly overexpressed member in human gastric cancer tissue samples (P < .001). We also detected high levels of Fgfr4 mRNA and protein in gastric dysplasia and adenocarcinoma lesions in mouse models. Infection with J166, 7.13, and PMSS1 cytotoxin-associated gene A (CagA)+ H pylori strains induced FGFR4 mRNA and protein expression in invitro and invivo models. This was associated with a concordant activation of signal transducer and activator of transcription 3 (STAT3). Analysis of the FGFR4 promoter suggested several putative binding sites for STAT3. Using chromatin immunoprecipitation assay and an FGFR-promoter luciferase reporter containing putative STAT3 binding sites and their mutants, we confirmed a direct functional binding of STAT3 on the FGFR4 promoter. Mechanistically, we also discovered a feedforward activation loop between FGFR4 and STAT3 where the fibroblast growth factor 19-FGFR4 axis played an essential role in activating STAT3 in a steroid receptor coactivator-dependent manner. Functionally, we found that FGFR4 protected against H pylori-induced DNA damage and cell death. Our findings demonstrated a link between infection, inflammation, and FGFR4 activation, where a feedforward activation loop between FGFR4 and STAT3 is established via steroid receptor coactivator in response to H pylori infection. Given the relevance of FGFR4 to the etiology and biology of gastric cancer, we propose FGFR4 as a druggable molecular vulnerability that can be tested in patients with gastric cancer.

OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage.

Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3(refs.2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNAoctamermotif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.

Aberrant CDK7 Activity Drives the Cell Cycle and Transcriptional Dysregulation to Support Multiple Myeloma Growth: An Attractive Molecular Vulnerability

Multiple myeloma (MM) cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We have previously shown that chemical and genetic perturbation of transcriptional regulator CDK7 significantly and selectively impacted MM cell growth and viability, supporting it as a pharmacologically relevant target for MM. Indeed, selective CDK7 inhibitor YKL-5-124 was active against a large panel of MM cell lines and primary MM cells, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs). The efficacy of YKL-5-124 was confirmed in vivo in several murine models of MM, including disseminated models.Gene expression analysis after CDK7 inhibition in several MM cell lines revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells, supporting CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient MM cells. Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs.To identify synthetically lethal targets and mechanisms of resistance to CDK7 inhibition, we performed a genome-wide CRISPR-Cas9 knockout screen in the MM1S cells treated with YKL-5-124 or DMSO. We found that BCL6, NFKBIA and B, TRAF2, TSC1 and CSNK2A1, a subunit of CK2, were top synthetically lethal hits; whereas deletion of RB1, SF3B3 and the DNA-binding transcriptional activator TADA2A that regulates RNA-pol II transcription, led to resistance to YKL-5-124. Molecular mechanisms of intrinsic and acquired resistance to CDK7 inhibition are now under investigation and will be presented.In conclusion, our study demonstrates CDK7 as an attractive molecular vulnerability in MM that can be exploited therapeutically alone or in combination. DisclosuresShirasaki: FIMECS: Consultancy. Chesi: Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Pfizer: Consultancy; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; FIMECS: Consultancy, Honoraria; Nurix: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; BMS: Research Funding; TEVA: Research Funding. Munshi: Novartis: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Adaptive Biotechnology: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy.

P-099: Perturbation of CDK7 and super-enhancer driven transcriptional programs synergistically halts multiple myeloma cell proliferation

<h3>Background</h3> With the deluge of available genomic data in recent years, it is becoming evident that MM cells are characterized by cell cycle dysregulation, epigenetic heterogeneity, and perturbation of the transcriptional landscape. We here elucidated the biological role of CDK7 and explored the functional consequence of its inhibition in MM using chemical and genetic approaches, including a recently reported selective CDK7 covalent inhibitor YKL-5-124, and engineered systems for rapid CDK7 protein degradation (dTAG). As previously shown with non-selective inhibitors, CDK7 inhibition via YKL-5-124 was active against a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. The efficacy of YKL-5-124 was confirmed in vivo in several murine MM models. Selective pharmacological degradation of endogenously tagged CDK7 and inducible KO/KD cell systems confirmed impact of CDK7 inhibition on MM cell proliferation supporting the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data support the notion of CDK7 as a central hub in the oncogenic CDK-pRb-E2F pathway in MM cells, with its expression and activity positively correlated with E2F transcriptional output in patient cells. <h3>Conclusion</h3> Importantly, dual inhibition with low doses of YKL-5-124 and BRD4 inhibitor JQ1 respectively, displayed superior activity against a panel of MM cell lines and primary MM cells compared to single perturbation alone by both converging on a subset of key SE-associated dependencies as well as impacting distinct oncogenic expression programs. Isobologram analysis revealed strong synergism with a combination index (CI) <1.0 at all tested doses, including in the cell lines with intrinsic resistance to YKL-5-124 or JQ1, while PBMC from healthy donors were less sensitive to the combination therapy In conclusion, our study demonstrates that CDK7 represents an attractive molecular vulnerability in MM to be exploited therapeutically alone or in combination.

Improving the therapeutic ratio of radiotherapy against radioresistant cancers: Leveraging on novel artificial intelligence-based approaches for drug combination discovery.

Despite numerous advances in cancer radiotherapy, tumor radioresistance remain one of the major challenges limiting treatment efficacy of radiotherapy. Conventional strategies to overcome radioresistance involve understanding the underpinning molecular mechanisms, and subsequently using combinatorial treatment strategies involving radiation and targeted drug combinations against these radioresistant tumors. These strategies exploit and target the molecular fingerprint and vulnerability of the radioresistant clones to achieve improved efficacy in tumor eradication. However, conventional drug-screening approaches for the discovery of new drug combinations have been proven to be inefficient, limited and laborious. With the increasing availability of computational resources in recent years, novel approaches such as Quadratic Phenotypic Optimization Platform (QPOP), CURATE.AI and Drug Combination and Prediction and Testing (DCPT) platform have emerged to aid in drug combination discovery and the longitudinally optimized modulation of combination therapy dosing. These platforms could overcome the limitations of conventional screening approaches, thereby facilitating the discovery of more optimal drug combinations to improve the therapeutic ratio of combinatorial treatment. The use of better and more accurate models and methods with rapid turnover can thus facilitate a rapid translation in the clinic, hence, resulting in a better patient outcome. Here, we reviewed the clinical observations, molecular mechanisms and proposed treatment strategies for tumor radioresistance and discussed how novel approaches may be applied to enhance drug combination discovery, with the aim to further improve the therapeutic ratio and treatment efficacy of radiotherapy against radioresistant cancers.

The functional GRHL3-filaggrin axis maintains a tumor differentiation potential and influences drug sensitivity

Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients’ data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.

41P The functional GRHL3-FLG axis predicts targeted therapy response in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease harbouring the most frequent hotspot mutations in the differentiation genes. Disruption of epithelial differentiation acts as a primary driver of HNSCC development and correlates with a poor patient's prognosis. To date, in addition to the non-selective conventional HNSCC treatments, such as chemotherapies and surgeries, the only FDA-approved targeted therapy Cetuximab, an epidermal growth factor receptor inhibitor, has a low response rate with considerable toxicity. Therefore, determining whether functional differentiation can serve as a molecular vulnerability and/or a predictor of targeted therapy in HNSCC is an area of valuable clinical need. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity screening was employed in tumours from a spontaneous HNSCC murine model, HNSCC patient’s tumours, and established human cell lines to reveal potential predictive biomarkers for targeted therapies. CRISPR-Cas9-mediated gene knockout and activation validated candidate predictors in HNSCC cell lines treated with inhibitors of the PI3K/mTOR, c-MYC and STAT3 pathways, the key signalling in HNSCC oncogenesis. We identified a novel Grainyhead-like 3 - Filaggrin (GRHL3-FLG) differentiation axis as a predictive biomarker to targeted therapy response in HNSCC. A subset of HNSCC with functional GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-MYC and STAT3 signaling. Furthermore, we identified the GRHL3 transcriptional target gene FLG as a novel tumour differentiation gene and, more importantly, stratified HNSCC subsets as treatment-resistant based on their FLG mutational profile. Moreover, the loss of FLG in sensitive HNSCC cells resulted in a dramatic resistance to targeted therapies while the GRHL3hi-FLGwt signature predicted a favourable patient's prognosis. Functional differentiation (GRHL3hi-FLGwt) provides the first example of differentiation-dependent therapy response and establishes a rationale for clinical investigation of differentiation-paired targeted therapy that may improve outcomes in HNSCC and other heterogeneous cancers.

Molecular mechanisms of neurodegeneration in the entorhinal cortex that underlie its selective vulnerability during the pathogenesis of Alzheimer's disease.

ABSTRACTThe entorhinal cortex (EC) is a vital component of the medial temporal lobe, and its contributions to cognitive processes and memory formation are supported through its extensive interconnections with the hippocampal formation. During the pathogenesis of Alzheimer's disease (AD), many of the earliest degenerative changes are seen within the EC. Neurodegeneration in the EC and hippocampus during AD has been clearly linked to impairments in memory and cognitive function, and a growing body of evidence indicates that molecular and functional neurodegeneration within the EC may play a primary role in cognitive decline in the early phases of AD. Defining the mechanisms underlying molecular neurodegeneration in the EC is crucial to determining its contributions to the pathogenesis of AD. Surprisingly few studies have focused on understanding the mechanisms of molecular neurodegeneration and selective vulnerability within the EC. However, there have been advancements indicating that early dysregulation of cellular and molecular signaling pathways in the EC involve neurodegenerative cascades including oxidative stress, neuroinflammation, glia activation, stress kinases activation, and neuronal loss. Dysfunction within the EC can impact the function of the hippocampus, which relies on entorhinal inputs, and further degeneration within the hippocampus can compound this effect, leading to severe cognitive disruption. This review assesses the molecular and cellular mechanisms underlying early degeneration in the EC during AD. These mechanisms may underlie the selective vulnerability of neuronal subpopulations in this brain region to the disease development and contribute both directly and indirectly to cognitive loss.This paper has an associated Future Leader to Watch interview with the first author of the article.

Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

A Unique Morphological Phenotype in Chemoresistant Triple-Negative Breast Cancer Reveals Metabolic Reprogramming and PLIN4 Expression as a Molecular Vulnerability.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

Abstract P4-07-01: A small molecule inhibitor (ERX-41) induces endoplasmic reticulum stress in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer and represents a disproportional share of the breast cancer mortality, primarily due to a lack of targeted therapies. There is a major unmet need for rationally designed novel therapies that can extend survival of patients with TNBC. TNBCs are characterized by a high basal level of endoplasmic reticulum stress, due to high protein turnover and need for proliferation. Recent studies revealed the role of several members of the Nuclear Receptor (NR) superfamily as molecular drivers in TNBC, including the androgen receptor (AR), glucocorticoid receptor (GR) and the orphan NR tailless (TLX). Methods: Recently, using peptidomimetics, we have developed small molecules that specifically target and block interactions of multiple coregulators with oncogenic NRs. We performed a screen of our 500+ compound peptidomimetic library derived from our ERX-11 oligobenzamide (that was rationally designed to target ERα) for anti-proliferative activity in TNBC cell lines. Identified leads were then validated in multiple TNBC cell lines. In vitro activity was tested using Cell titer glo, MTT, matrigel invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, CRISPR/Cas9 KO and RNA-seq analysis. Xenograft, patient derived xenograft (PDX), patient derived explant (PDE) and xenograft derived explant (XDE) TNBC models were used for preclinical evaluation and toxicity. Results: We have identified a first-in-class drug (ERX-41) that has potent activity (IC50 = 50-250nM) against all six molecular subtypes of TNBC. Systematic evaluation using CRISPR/Cas9 KO screen and overexpression screen comprising 48 NRs identified TLX as a preferred target of ERX-41. Analyses of primary breast tumors revealed TLX was highly expressed in TNBC. Further, TLX was amplified in nearly 50% of TNBC xenografts (cbioportal.org). Modelling, mechanistic and biochemical studies showed that ERX-41 interact with TLX and selectively blocks its interactions with coregulators. Gene expression analyses revealed both significant reduction of TLX-activated genes (CCND1, WNT7A) and significant activation of TLX-repressed genes (p21) upon treatment with ERX-41 in TNBC models. Gene ontogeny pathway analyses of RNA-seq data in TNBC cells showed that ERX-41 treatment positively correlated with apoptosis. Our ultrastructural studies indicated that ERX-41 enhances endoplasmic reticulum stress in TNBC inducing autophagic flux and subsequent apoptosis. ERX-41 has significant potency against multiple TNBC xenografts and PDXs in vivo, PDEs and XDEs ex vivo, indicating its potential for clinical translation. Pharmacologically, ERX-41 exhibited high oral bioavailability and associated with minimal toxicity upon oral gavage for up to 120 days in animal studies. Conclusions: We believe that the ability of ERX-41 to block NR signaling and target a critical molecular vulnerability in TNBC and its ability to enhance endoplasmic reticulum stress in TNBC, will revolutionize the therapeutic landscape of TNBC. ERX-41 is oral bioavailable, potent against multiple TNBC molecular subtypes, and is associated with minimal systemic side effects. (supported by NIH grant RO1 CA223828-01) Citation Format: Liu X, Viswanadhapalli S, Ma S, Lee T-K, Sareddy GR, Ekoue DN, Blatt EM, Zhou M, Li M, Tekmal RR, Ahn J-m, Vadlamudi RK, Raj GV. A small molecule inhibitor (ERX-41) induces endoplasmic reticulum stress in triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-01.

Inhibition of MYC attenuates tumor cell self-renewal and promotes senescence in SMARCB1-deficient Group 2 atypical teratoid rhabdoid tumors to suppress tumor growth in vivo.

Loss of SMARCB1 is the hallmark genetic event that characterizes rhabdoid tumors in children. Rhabdoid tumors of the brain (ATRT) occur in young children and are particularly challenging with poor long-term survival. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. Recent studies demonstrate that ATRT consists of 3 genomic subgroups with a subset of poor outcome tumors expressing high BMP and MYC pathway activation. Here we show that MYC occupies distinct promoter loci in ATRT compared to embryonic stem (ES) cells. Furthermore, using human ATRT cell lines, patient-derived cell culture, ex vivo patient-derived tumor, and orthotopic xenograft models, we show that MYC inhibition is a molecular vulnerability in SMARCB1-deleted tumors and that such inhibition effectively suppresses BMP and pluripotency-associated genomic programs, attenuates tumor cell self-renewal, promotes senescence, and inhibits ATRT tumor growth in vivo. Transgenic expression of Omomyc (a bona-fide MYC dominant negative) or chemical inhibition of MYC transcriptomic programs with the BET inhibitor JQ1 phenocopy genetic depletion of MYC, effectively restricting ATRT tumor growth and opening a promising therapeutic avenue for rhabdoid tumors in children.

Effect of Exercise and Aβ Protein Infusion on Long-Term Memory-Related Signaling Molecules in Hippocampal Areas.

Alzheimer's disease (AD) results from over-production and aggregation of β-amyloid (Aβ) oligopeptides in the brain. The benefits of regular physical exercise are now recognized in a variety of disorders including AD. In order to understand the effect of exercise at the molecular level, we studied the impact of exercise on long-term memory-related signaling molecules in an AD rat model. The rat model of AD (AD rat) was produced by 14-day osmotic pump infusion of i.c.v. 250pmol/day Aβ1-42. The effects of 4weeks of regular rodent treadmill exercise on the protein levels of CREB, CaMKVI, and MAPK-ERK1/2 in this model were determined by immunoblot analysis in the CA1 and dentate gyrus (DG) areas of the hippocampus, which is among the first brain structures impacted by AD. Aβ infusion caused marked reductions in the basal protein levels of CaMKVI and phosphorylated CREB without significantly affecting total CREB levels in both CA1 and DG areas. As predicted, our exercise regimen totally prevented these effects in the brains of exercised AD rats. Surprisingly, however, neither Aβ infusion nor exercise had any significant effect on the levels of phosphorylated or total ERK in the CA1 and DG areas. Additionally, exercise did not increase any of these molecules in healthy normal rats, which indicated a protective effect of exercise. These findings suggest that CaMKIV is likely a major kinase for phosphorylation of CREB. Therefore, regular exercise is highly effective in preventing the effects of AD even at the molecular level in both areas of the hippocampus. Considering the well-known resistance of the DG area to insults relative to area CA1, the present findings revealed similar molecular vulnerability of the two areas to AD pathology.

Abstract B03: Targeting the KIF11/KIF15/TPX2 axis to develop new therapies for ovarian cancer

Abstract Unlimited genetic and epigenetic diversity across the patient population, and within the tumor and among metastatic lesions of an individual patient, presents a challenge in developing targeted therapies to treat epithelial ovarian cancer (EOC). To find the Achilles heel of ovarian cancers, we designed an RNAi-based screen (&amp;gt;6,000 genes) to identify points of molecular vulnerability. These screens identified KIF11 as essential to maintain tumor cell viability. KIF11 encodes kinesin Eg5 (KIF11), a motor protein required for microtubule antiparallel sliding during mitosis that has been targeted clinically. Although KIF11 inhibitors are well tolerated by EOC patients, clinical response rates were disappointing in heavily treated patients with advanced disease. We hypothesize one mechanism through which KIF11 inhibitors’ efficacy is muted is via functional compensation of mitotic spindle assembly by KIF15, a second motor kinesin. Using a synthetic lethal approach, we have shown that RNAi-mediated silencing of KIF15 has no effect on cell viability, but significantly sensitized cells by &amp;gt;10- to 100-fold to a class of KIF11 inhibitors (KIF11is). TPX2-targeted siRNAs reduce cell viability by 5-fold alone, and sensitize cells by 100-fold in the presence of KIF11i. Based on these observations, we designed a high-throughput screening assay using Alpha Technology©. Informed by previous predictions of KIF15-TPX2 binding domains, we designed peptides corresponding to the appropriate domains of KIF15 (aa1149-1388, GST-tagged) and TPX2 (aa346-747, His-tagged), for use in the AlphaScreen© assay. Following several optimization steps (e.g., buffer selection, protein titration, TruHits© assay validation), we demonstrate that KIF15 binds TPX2, producing a signal &amp;gt;30-fold greater than background signal. Further, peptides designed to mimic either KIF15 or TPX2 binding domains, but not scrambled controls, exhibited concentration-dependent disruption of the KIF15-TPX2 interaction. Next, we screened a FDA library (3,474 compounds) and bioactive (1,902 compounds) library and identified 122 hits; fresh compound stocks were tested at 4 concentrations (20, 10, 5, and 2.5 μM), resulting in 53 confirmed hits (28 FDA, 25 bioactive). To identify false positives that interfere with assay chemistry, we repeated the screen at 3 concentrations, replacing the His-TPX2 and GST-KIF15 proteins with a His-GST fusion. As an orthogonal screen, we performed a Homogenous Time Resolved Fluorescence© (HTRF)-based screen to reconfirm hit compounds at the aforementioned concentrations. From these combined data, we have identified 4 hit compounds (KU-P001 to KU-P004) that disrupt KIF15/TPX2 protein-protein interactions. To further prioritize our hits, we developed in vitro (EOC cell lines were exposed to increasing concentrations, up to 1 µM, of KIF11i for 4 months) and in vivo (patient-derived ascites xenograft models of EOC were treated with 3 cycles of either 5 mg/kg or 10 mg/kg of a KIF11i and were serially passaged and treated for 3 additional generations) models of KIF11i-resistance to evaluate their activities. In resistant EOC cells we observed a 3-fold increase in KIF15 protein expression as compared with parental cells; TPX2 protein expression remained unchanged. In vivo, both KIF15 (68% increase) and TPX2 (2-fold) protein expression was increased in resistant versus untreated PDX tumor cells. Combination studies of KU-P004 and ispinesib (KIF11i) in vitro revealed a 2- to 64-fold increase in cell death for both responsive and resistant EOC cells compared with KIF11i treatment alone. In summary, we have developed a high-throughput screening assay platform to identify inhibitors of the KIF15-TPX2 interaction and are using iterative medicinal chemistry to guide optimization and functional evaluation of hits and lead compounds to develop new combination therapies for women with ovarian cancer. Citation Format: Rebecca J. Wates, Anuradha Roy, Frank Schoenen, John Karanicolas, Scott Weir, Andrew Godwin. Targeting the KIF11/KIF15/TPX2 axis to develop new therapies for ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B03.

The \u03b220\u2013\u03b221 of gp120 is a regulatory switch for HIV-1 Env conformational transitions

The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins (Env). Binding to the CD4 receptor triggers a cascade of conformational changes in distant domains that move Env from a functionally “closed” State 1 to more “open” conformations, but the molecular mechanisms underlying allosteric regulation of these transitions are still elusive. Here, we develop chemical probes that block CD4-induced conformational changes in Env and use them to identify a potential control switch for Env structural rearrangements. We identify the gp120 β20–β21 element as a major regulator of Env transitions. Several amino acid changes in the β20–β21 base lead to open Env conformations, recapitulating the structural changes induced by CD4 binding. These HIV-1 mutants require less CD4 to infect cells and are relatively resistant to State 1-preferring broadly neutralizing antibodies. These data provide insights into the molecular mechanism and vulnerability of HIV-1 entry.

Mycobacterium abscessus l,d-Transpeptidases Are Susceptible to Inactivation by Carbapenems and Cephalosporins but Not Penicillins.

As a growing number of clinical isolates of Mycobacterium abscessus are resistant to most antibiotics, new treatment options that are effective against these drug-resistant strains are desperately needed. The majority of the linkages in the cell wall peptidoglycan of M. abscessus are synthesized by nonclassical transpeptidases, namely, the l,d-transpeptidases. Emerging evidence suggests that these enzymes represent a new molecular vulnerability in this pathogen. Recent studies have demonstrated that inhibition of these enzymes by the carbapenem class of β-lactams determines their activity against Mycobacterium tuberculosis Here, we studied the interactions of β-lactams with two l,d-transpeptidases in M. abscessus, namely, LdtMab1 and LdtMab2, and found that both the carbapenem and cephalosporin, but not penicillin, subclasses of β-lactams inhibit these enzymes. Contrary to the commonly held belief that combination therapy with β-lactams is redundant, doripenem and cefdinir exhibit synergy against both pansusceptible M. abscessus and clinical isolates that are resistant to most antibiotics, which suggests that dual-β-lactam therapy has potential for the treatment of M. abscessus Finally, we solved the first crystal structure of an M. abscessus l,d-transpeptidase, LdtMab2, and using substitutions of critical amino acids in the catalytic site and computational simulations, we describe the key molecular interactions between this enzyme and β-lactams, which provide an insight into the molecular basis for the relative efficacy of different β-lactams against M. abscessus.

HILAQ: A Novel Strategy for Newly Synthesized Protein Quantification.

Here we describe a new strategy, HILAQ (Heavy Isotope Labeled Azidohomoalanine Quantification), to rapidly quantify the molecular vulnerability profile to oxytosis, which is an oxidative stress-induced programed cell death pathway that has been reported to be involved in aging and neurodegenerative diseases. HILAQ was able to quantify 1962 newly synthesized proteins (NSPs) after 1 h of pulse labeling in HEK293T cell line, while 353 proteins were quantified using the previously published QuaNCAT protocol. HILAQ was successfully applied to the HT22 oxytosis model. 226 proteins were found to have a two-fold change in abundance, and 108 proteins were enriched in the cell death pathway, demonstrating the utility of HT22 cells as a tool to study the molecular details of cell death involved in neurodegenerative diseases. The HILAQ strategy simplifies the analysis of newly synthesized proteomes through the use of isobaric labels and achieves higher sensitivity than previously published methods.