Lethal Screen Research Articles

SIK2 kinase synthetic lethality is driven by spindle assembly defects in FANCA-deficient cells.

The Fanconi anemia (FA) pathway safeguards genomic stability through cell cycle regulation and DNA damage repair. The canonical tumor suppressive role of FA proteins in the repair of DNA damage during interphase is well established, but their function in mitosis is incompletely understood. Here, we performed a kinome‐wide synthetic lethality screen in FANCA −/− fibroblasts, which revealed multiple mitotic kinases as necessary for survival of FANCA‐deficient cells. Among these kinases, we identified the depletion of the centrosome kinase SIK2 as synthetic lethal upon loss of FANCA. We found that FANCA colocalizes with SIK2 at multiple mitotic structures and regulates the activity of SIK2 at centrosomes. Furthermore, we found that loss of FANCA exacerbates cell cycle defects induced by pharmacological inhibition of SIK2, including impaired G2‐M transition, delayed mitotic progression, and cytokinesis failure. In addition, we showed that inhibition of SIK2 abrogates nocodazole‐induced prometaphase arrest, suggesting a novel role for SIK2 in the spindle assembly checkpoint. Together, these findings demonstrate that FANCA‐deficient cells are dependent upon SIK2 for survival, supporting a preclinical rationale for targeting of SIK2 in FA‐disrupted cancers.

Splitting the Functions of a Mitochondrial Iron/Pyrimidine Carrier

The biosynthesis of the iron-cofactors heme and iron-sulfur [Fe-S] clusters takes place in the interior of the mitochondria, but the mechanism of delivery of iron across the impermeable inner mitochondrion membrane is still unclear. In Saccharomyces cerevisiae the mitochondrial carrier proteins Mrs3 and Mrs4, are involved in iron transport, as is the human homolog mitoferrin 1, but yeast lacking these genes still have normal mitochondrial iron content, suggesting involvement of other proteins. A synthetic lethal screen identified a requirement of Rim2 in the absence of Mrs3 and Mrs4. Rim2 of S. cerevisiae, and the human homologs SLC25A33 and SLC25A36, import and export pyrimidine nucleotides in and out of mitochondria. Rim2 also delivers iron into intact mitochondria, allowing conversion of a fluorescent porphyrin precursor into non-fluorescent heme inside the organelle. Measurement of [Fe-S] assembly, using 35S-cysteine, and loading into the aconitase, demonstrated that Rim2 mediates mitochondrial iron uptake for [Fe-S] cluster biogenesis.Point mutations were generated in the predicted substrate binding site of Rim2. K299 on helix 5 and E248 on helix 4 showed asymmetry and were predicted to be substrate binding residues, and these were mutated respectively to alanine. In intact mitochondria, incorporation of radiolabeled iron (55Fe) into heme, was significantly decreased in the Rim2-E248A mutant. Additionally protein levels of aconitase in the Rim2-E248 mutant were much lower, suggestive of a destabilization of aconitase in the absence of [Fe-S] cluster incorporation. Interestingly, TTP (pyrimidine) homoexchange was entirely preserved in this mutant. The TTP/TMP heteroexchange was compromised, perhaps because the E248 residue was required for proton coupled transport. Conversely for the Rim2-K299A mutant, all pyrimidine exchange functions were severely compromised almost to the point of the Rim2 null. By contrast, iron dependent functions, such as 55Fe transport and incorporation into heme, were maintained at wild-type levels in the K299A mutant.In summary the lysine at position 299 of Rim2 was critical for pyrimidine transport/exchange where as the glutamic acid at position 248 was required for iron transport/use and proton coupled TTP/TMP heteroexchange across the mitochondrial inner membrane. Iron transport by Rim2 is likely to be proton coupled and pyrimidine independent. DisclosuresNo relevant conflicts of interest to declare.

Direct P70S6K1 inhibition to replace dexamethasone in synergistic combination with MCL-1 inhibition in multiple myeloma

AbstractNovel combination therapies have markedly improved the lifespan of patients with multiple myeloma (MM), but drug resistance and disease relapse remain major clinical problems. Dexamethasone and other glucocorticoids are a cornerstone of conventional and new combination therapies for MM, although their use is accompanied by serious side effects. We aimed to uncover drug combinations that act in synergy and, as such, allow reduced dosing while remaining effective. Dexamethasone and the myeloid cell leukemia 1 (MCL-1) inhibitor S63845 (MCL-1i) proved the most potent combination in our lethality screen and induced apoptosis of human myeloma cell lines (HMCLs) that was 50% higher compared with an additive drug effect. Kinome analysis of dexamethasone-treated HMCLs revealed a reduction in serine/threonine peptide phosphorylation, which was predicted to result from reduced Akt activity. Biochemical techniques showed no dexamethasone-induced effects on FOXO protein or GSK3 but did show a 50% reduction in P70S6K phosphorylation, downstream of the Akt-mTORC1 axis. Replacing dexamethasone by the P70S6K1 isoform-specific inhibitor PF-4708671 (S6K1i) revealed similar and statistically significant synergistic apoptosis of HMCLs in combination with MCL-1i. Interestingly, apoptosis induced by the P70S6K1i and MCL-1i combination was more-than-additive in all 9 primary MM samples tested; this effect was observed for 6 of 9 samples with the dexamethasone and MCL-1i combination. Toxicity on stem and progenitor cell subsets remained minimal. Combined, our results show a strong rationale for combination treatments using the P70S6K inhibitor in MM. Direct and specific inhibition of P70S6K may also provide a solution for patients ineligible or insensitive to dexamethasone or other glucocorticoids.

HGG-33. EXPLOITING METABOLIC DEFECTS WITH NAMPT INHIBITORS IN DIPG

Diffuse intrinsic pontine glioma (DIPG) are universally lethal pediatric brain tumors with limited treatment options. We recently performed synthetic lethal drug screen with a panel of DNA repair and metabolic inhibitors in vitro, in patient-derived DIPG cells and isogenic cell lines engineered to contain key DIPG-associated mutations. Nearly 80% of DIPGs harbor a recurrent H3K27M mutation in H3.3 (H3F3A) or H3.1 (HIST1H3B) histones. This has prompted us to consider H3K27M mutation-induced exploitable defects for a therapeutic gain. This screen identified synthetic lethal interactions between H3K27M mutations and the nicotinamide phosphoribosyl transferase (NAMPT) inhibitor, FK866. The association between H3K27M mutations and NAMPTi sensitivity was validated in follow-up studies using isogenic WT and H3K27M-mutant expressing pairs of human immortalized astrocytes and neural progenitor cells (NPCs). In addition, we tested the effects of FK866 in a panel of unique DIPG patient-derived tumor neurospheres in short-term viability assays. We found that FK866 induced depletion of NAD and exhibited toxicity towards H3K27M mutant DIPG cell lines with an IC50 of ~2.5 nM. These findings were consistent across three structurally unique NAMPT inhibitors. Finally, we found that inducible expression of mutant H3K27M results in a gradual, 50% decrease in cellular NAD levels over the course of five days, suggesting that H3K27M mutations may induce an inherent NAD metabolic defect that is exploited by NAMPTi’s. We now seek to understand the mechanistic basis for the observed metabolic defect and NAMPTi sensitivity associated with mutant H3K27M. In addition, we aim to identify specific NAMPTi and DNA damaging agent combinations which maximally exploit this H3K27M-associated NAD metabolic defects.

DDRE-29. DE NOVO PYRIMIDINE SYNTHESIS IS A TARGETABLE VULNERABILITY IN IDH-MUTANT GLIOMA

70–90% of lower-grade gliomas and secondary glioblastomas harbor gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1), causing overproduction of the oncometabolite (R)-2-hydroxyglutarate [(R)-2HG]. Although inhibitors of mutant IDH enzymes are effective in other cancers, including leukemia, they have shown guarded efficacy in preclinical and clinical brain tumor studies, thus underscoring the need to identify additional therapeutic targets in IDH mutant glioma. We sought to identify tumor-specific metabolic vulnerabilities induced by IDH1 mutations that could be exploited therapeutically. To uncover such vulnerabilities, we conducted a chemical synthetic lethality screen using isogenic IDH1 mutant and IDH1 wild-type (WT) glioma cell lines and a novel metabolic inhibitor screening platform. We discovered that IDH1 mutant cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). This vulnerability is specific because inhibitors of purine nucleotide metabolism did not score in our screen. We validated that the cytotoxicity of pyrimidine synthesis inhibitors is on-target and showed that IDH1 mutant patient-derived glioma stem-like cell lines are also hyperdependent on de novo pyrimidine nucleotide synthesis compared to IDH1 WT lines. To test pyrimidine synthesis dependence of IDH1 mutant gliomas in vivo, we used a brain-penetrent DHODH inhibitor currently undergoing evaluation in leukemia patients, BAY 2402234. We found that BAY 2402234 displays monotherapy activity against gliomas in an orthotopic xenograft model of IDH1 mutant glioma, with an effect size that compared favorably with radiotherapy. We also developed novel genetically engineered and allograft mouse models of mutant IDH1-driven anaplastic astrocytoma and showed that BAY 2402234 blocked growth of orthotopic astrocytoma allografts. Our findings bolster rationale to target DHODH in glioma, highlight BAY 2402234 as a clinical-stage drug that can be used to inhibit DHODH in brain tumors, and establish IDH1 mutations as predictive biomarkers of DHODH inhibitor efficacy.

Therapeutic Escape in Gαq-mutant Uveal Melanoma: It's a FAK.

Through a synthetic lethal screen, ERK activation was found to mediate resistance to FAK inhibition in GNAQ-mutant uveal melanoma. With PLCB-PKC-ERK and Trio-FAK-Yap representing compensatory effectors of mutant Gαq signaling, combined inhibition of both pathways may be a promising therapeutic strategy in metastatic uveal melanoma.See related article by Paradis et al., p. 3190.

Inhibition of ERAD synergizes with FTS to eradicate pancreatic cancer cells

BackgroundPancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is driven by oncogenic KRAS mutations. Farnesyl thiosalicylic acid (FTS), also known as salirasib, is a RAS inhibitor that selectively dislodges active RAS proteins from cell membrane, inhibiting downstream signaling. FTS has demonstrated limited therapeutic efficacy in PDAC patients despite being well tolerated.MethodsTo improve the efficacy of FTS in PDAC, we performed a genome-wide CRISPR synthetic lethality screen to identify genetic targets that synergize with FTS treatment. Among the top candidates, multiple genes in the endoplasmic reticulum-associated protein degradation (ERAD) pathway were identified. The role of ERAD inhibition in enhancing the therapeutic efficacy of FTS was further investigated in pancreatic cancer cells using pharmaceutical and genetic approaches.ResultsIn murine and human PDAC cells, FTS induced unfolded protein response (UPR), which was further augmented upon treatment with a chemical inhibitor of ERAD, Eeyarestatin I (EerI). Combined treatment with FTS and EerI significantly upregulated the expression of UPR marker genes and induced apoptosis in pancreatic cancer cells. Furthermore, CRISPR-based genetic ablation of the key ERAD components, HRD1 and SEL1L, sensitized PDAC cells to FTS treatment.ConclusionOur study reveals a critical role for ERAD in therapeutic response of FTS and points to the modulation of UPR as a novel approach to improve the efficacy of FTS in PDAC treatment.

Targeting "undruggable" c-Myc protein by synthetic lethality.

Synthetic lethal screening, which exploits the combination of mutations that result in cell death, is a promising method for identifying novel drug targets. This method provides a new avenue for targeting "undruggable" proteins, such as c-Myc. Here, we revisit current methods used to target c-Myc and discuss the important functional nodes related to c-Myc in non-oncogene addicted network, whose inhibition may cause a catastrophe for tumor cell destiny but not for normal cells. We further discuss strategies to identify these functional nodes in the context of synthetic lethality. We review the progress and shortcomings of this research field and look forward to opportunities offered by synthetic lethal screening to treat tumors potently.

Synthetic Lethal Screens Reveal Cotargeting FAK and MEK as a Multimodal Precision Therapy for GNAQ-Driven Uveal Melanoma.

Uveal melanoma is the most common eye cancer in adults. Approximately 50% of patients with uveal melanoma develop metastatic uveal melanoma (mUM) in the liver, even after successful treatment of the primary lesions. mUM is refractory to current chemo- and immune-therapies, and most mUM patients die within a year. Uveal melanoma is characterized by gain-of-function mutations in GNAQ/GNA11, encoding Gαq proteins. We have recently shown that the Gαq-oncogenic signaling circuitry involves a noncanonical pathway distinct from the classical activation of PLCβ and MEK-ERK. GNAQ promotes the activation of YAP1, a key oncogenic driver, through focal adhesion kinase (FAK), thereby identifying FAK as a druggable signaling hub downstream from GNAQ. However, targeted therapies often activate compensatory resistance mechanisms leading to cancer relapse and treatment failure. We performed a kinome-wide CRISPR-Cas9 sgRNA screen to identify synthetic lethal gene interactions that can be exploited therapeutically. Candidate adaptive resistance mechanisms were investigated by cotargeting strategies in uveal melanoma and mUM in vitro and in vivo experimental systems. sgRNAs targeting the PKC and MEK-ERK signaling pathways were significantly depleted after FAK inhibition, with ERK activation representing a predominant resistance mechanism. Pharmacologic inhibition of MEK and FAK showed remarkable synergistic growth-inhibitory effects in uveal melanoma cells and exerted cytotoxic effects, leading to tumor collapse in uveal melanoma xenograft and liver mUM models in vivo. Coupling the unique genetic landscape of uveal melanoma with the power of unbiased genetic screens, our studies reveal that FAK and MEK-ERK cotargeting may provide a new network-based precision therapeutic strategy for mUM treatment.See related commentary by Harbour, p. 2967.

The Replication Stress Response Kinase ATR is an Interferon Signaling Driven Co-Dependency In Pancreas Cancer Cells

An inflammatory, cytokine-rich tumor microenvironment is a defining feature of pancreatic ductal adenocarcinoma (PDAC). Among proinflammatory cytokines, type I interferons (IFNs) are particularly important as they regulate the expression of hundreds of genes. It is however unclear whether IFN signaling in PDAC tumors can be leveraged therapeutically. We determined that a type I IFN response transcriptional signature is enriched in a subset of PDAC and that IFN signaling biomarkers are detectable in patient-derived and cell line xenograft tumors. To systematically identify actionable vulnerabilities induced by IFN, we integrated a mass spectrometry-based phosphoproteomic analysis and a chemical genomics high-throughput synthetic lethality screen which together revealed that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) and sensitizes tumor cells to ATR inhibitors. In vitro, IFNs induce replication stress and cell cycle arrest in S-phase which is accompanied by nucleotide pool insufficiency and enhanced nucleoside efflux into the extracellular environment. In combination with IFN, ATR inhibitors induce lethal DNA damage, downregulate de novo nucleotide biosynthesis mediated by ribonucleotide reductase, and deplete nucleotide pools. In vivo, ATR inhibitors limit the growth of subcutaneous and orthotopic PDAC tumors in which IFN production is driven by the activation of stimulator of interferon genes (STING). These results highlight critical crosstalk between IFN signaling, the DNA replication stress response network, and nucleotide metabolism in PDAC while providing the rationale for exploring targeted therapeutic interventions that leverage the tumor cell autonomous effects of IFN signaling.

A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae.

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5′ region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

DIPG-79. H3K27M INDUCES EPIGENETIC AND ONCOGENIC CHANGES THAT ARE PARTIALLY REVERSED BY SMALL MOLECULE AURORA KINASE B/C INHIBITION

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor with no curative treatments. Approximately 80% of DIPGs contain an H3K27M mutation. The implications of the mutation and how they may be targeted are not fully understood. We established an H3K27M effect-isolating model by transducing H3K27-wildtype lines (HSJD-GBM-001, normal human astrocytes) with lentiviral-packaged H3K27M. We characterized H3K27M-related changes through western blot, phenotypic assays, and RNA-seq. Drug screening of H3K27-wildtype and matched H3K27M-transduced lines was used to identify targets more effective with H3K27M present. Patient-derived pediatric glioblastoma and DIPG lines (BT-245, SU-DIPG-IV, HSJD-DIPG-007, SU-DIPG-XIII*, SF7761) were used for validation. We observed increased H3K27ac and decreased H3K27me3, as well as increased proliferative and migratory abilities, with the addition of H3K27M to H3K27-wildtype lines. RNA-seq showed downregulation of cell cycle regulation and upregulation of epithelial-mesenchymal transition. GSK1070916, an Aurora kinase B/C inhibitor, was isolated from a synthetic lethality screen with H3K27M. GSK1070916 showed strong efficacy in native H3K27M lines (IC50s=60nM-1250nM), superior to the Aurora kinase A inhibitor alisertib, to which all cell lines showed substantial resistance. Combination of both drugs was not synergistic. GSK1070916 treatment caused increased H3K27me3 and decreased H3S10ph and H3S28ph. GSK1070916 induced apoptosis and S-phase stall. The H3K27M mutation induces epigenetic, phenotypic, and cell cycle regulation changes resulting in relaxation of transcriptional controls and more aggressive growth. Aurora kinase B/C inhibition is a novel therapeutic modality for DIPG that appears capable of reversing some H3K27M-related epigenetic changes, inducing apoptosis, and repressing uncontrolled cellular division.

The E3 ligase UBR2 regulates cell death under caspase deficiency via Erk/MAPK pathway

Escape from cell death is a key event in cancer establishment/progression. While apoptosis is often considered as the main cell death pathway, upon caspase inhibition, cell death is rather delayed than blocked leading to caspase-independent cell death (CICD). Although described for years, CICD’s underlying mechanism remains to be identified. Here, we performed a genome-wide siRNA lethality screening and identified the RING-Type E3 Ubiquitin Transferase (UBR2) as a specific regulator of CICD. Strikingly, UBR2 downregulation sensitized cells towards CICD while its overexpression was protective. We established that UBR2-dependent protection from CICD was mediated by the MAPK/Erk pathway. We then observed that UBR2 is overexpressed in several cancers, especially in breast cancers and contributes to CICD resistance. Therefore, our work defines UBR2 as a novel regulator of CICD, found overexpressed in cancer cells, suggesting that its targeting may represent an innovative way to kill tumor cells.

DDRE-06. CELLULAR STRESS RESPONSE IN DIPG THERAPY

Abstract Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor of the pons and brainstem. Therefore, there is a desperate need for new therapeutics. Genomic profiling of tumors identified a highly prevalent dominant negative somatic mutation at lysine (K)-27 in histone genes HIST1H3B and H3F3A. Clonal evolution modeling suggests these mutations are truncal, and studies have demonstrated their contribution to tumorigenesis. ONC201, a first-in-class DRD2 antagonist and ClpP agonist is an anticancer drug developed by Oncoceutics, which targets the unfolded protein response (UPR) and integrated stress response (ISR) signaling and is actively being investigated in patients with recurrent H3 K27M-mutant gliomas. In adults with recurrent glioma, single agent studies showed benign-safety, no dose-limiting toxicities and a durable objective response when administered orally. In addition, intra-tumoral drug levels exceeded therapeutic thresholds, and induced tumor cell apoptosis. Based on this and response seen in a pediatric patient with DIPG for whom compassionate use of ONC201 was approved, a multi-arm, non-randomized multi-institutional Phase I clinical trial (NCT03416530) is actively accruing patients. However, the strength of UPR and ISR in DIPGs and their effect on DIPG response to ONC201 is not known. Our group employed bulk/single cell transcriptomic and single cell proteomic approaches to demonstrate substantial heterogeneity in UPR and ISR signaling in human DIPG samples. Consistent with this, DIPG cell lines exhibited considerable variability in sensitivity to ONC201. Single cell profiling identified tumor sub-populations with significant proliferative capacity even after ONC201 exposure. Incomplete response promotes recurrence. To target these cells, we performed a synthetic lethality screen with a library of 360 FDA-approved CNS penetrant compounds, which identified HDAC inhibitors and DNA damage-inducing chemotherapy as having synergy with ONC201. Thus, we suggest that tumor heterogeneity impacts sensitivity to ONC201 and that this can be reduced by combination treatments.

A Synthetic Lethal Approach to Eradicate AML Via Synergistic Activation of Pro-Apoptotic p53 By MDM2 and BET Inhibitors

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually compared to many other cancers, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation via MDM2i and minimize toxicity, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. These screens identified BRD4 inhibition as a candidate synthetic lethal partner of MDM2i. BRD4 is a member of the Bromodomains and Extraterminal (BET) family of proteins, a transcriptional co-activator and already a candidate AML therapeutic target. Surprisingly, we found inhibition of BRD4 alone induces expression of some of p53 target genes. We unexpectedly reveal that BRD4 binds to p53 target genes and acts as a transcriptional repressor of these genes. Synergistic cell killing by the drug combination (MDM2i + BET inhibitor (BETi)) depends on synergistic activation of p53 target genes, such as PUMA and NOXA, due to simultaneous stabilization of p53 by MDM2i and relief of BRD4-mediated repression by BETi. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type TP53in vitro, against two mouse models of AML in vivo, and against primary human patient blasts in vitro. Furthermore, we used BET PROTACs to selectively and completely induce degradation of BRD4 in cells. Consistent with results from BETi, BET degraders and MDM2i synergize to suppress cell viability with superior potency. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, co-targeting wild-type TP53 and a transcriptional repressor function of BRD4 represent a novel synthetic lethal vulnerability in AML. Disclosures Latif: AbbVie: Consultancy, Honoraria; Takeda UK: Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Copland:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Roche: Research Funding; Epizyme: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; Gilead: Speakers Bureau.

Inhibition of O-GlcNAc Transferase Renders Prostate Cancer Cells Dependent on CDK9.

O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed a specific and potent inhibitor targeting this enzyme, and here, we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II to promote transcription elongation. We show that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from patients with metastatic prostate cancer, but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells, but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer-specific vulnerabilities. IMPLICATIONS: The primary contribution of OGT to cell proliferation is unknown, and in this study, we used a compound screen to indicate that OGT and CDK9 collaborate to sustain a cancer cell-specific pro-proliferative program. A better understanding of how OGT and CDK9 cross-talk will refine our understanding of this novel synthetic lethal interaction.

Targeting the Synthetic Vulnerability of PTEN-Deficient Glioblastoma Cells with MCL1 Inhibitors.

PTEN deletion or mutation occurs in 30% to 60% of patients with glioblastoma (GBM) and is associated with poor prognosis. Efficacious therapy for this subgroup of patients is currently lacking. To identify potential target(s) to selectively suppress PTEN-deficient GBM growth, we performed a three-step synthetic lethal screen on LN18 PTEN wild-type (WT) and knockout (KO) isogeneic GBM cell lines using a library containing 606 target-selective inhibitors. A MCL1 inhibitor UMI-77 identified in the screen exhibited excellent suppression on the proliferation, colony formation, 3D spheroid, and neurosphere formation of PTEN-deficient GBM cells. Mechanistically, loss of PTEN in GBM cells led to upregulation of MCL1 in posttranslational level via inhibition of GSK3β, and consequently confer cells resistance to apoptosis. Pharmacologic inhibition or knockdown of MCL1 blocked this PI3K-GSK3β-MCL1 axis and caused reduction of several antiapoptotic proteins, finally induced massive caspase-3 cleavage and apoptosis. In both subcutaneous and orthotopic GBM models, knockdown of MCL1 significantly impaired the in vivo growth of PTEN-deficient xenografts. Moreover, the combination of UMI-77 and temozolomide synergistically killed PTEN-deficient GBM cells. Collectively, our work identified MCL1 as a promising target for PTEN-deficient GBM. For future clinical investigations, priority should be given to the development of a selective MCL1 inhibitor with efficient brain delivery and minimal in vivo toxicity.

Abstract 1897: Mechanism of MEK inhibitor resistance in triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive disease associated with recurrence, metastasis, and resistance to chemotherapy and radiation; effective targeted therapies are needed. Although the MEK inhibitors (MEKi) selumetinib (AZD6244) and pimasertib (AS703026) showed therapeutic efficacy in preclinical models, they failed as effective single agents in the clinic due to development of intrinsic resistance. To identify potential mediators of this resistance, we performed a synthetic lethal siRNA screen and identified myeloid cell leukemia-1 (MCL1) as a potential mediator of selumetinib resistance. Mcl-1 is an anti-apoptotic protein that is highly amplified in numerous human cancers and associated with cell immortalization and chemoresistance. We hypothesized that Mcl-1 promotes MEKi resistance in TNBC, and sought to confirm this and discern of mechanisms. Results: We established selumetinib- and pimasertib-resistant clones of TNBC cells SUM-149 and MDA-MB-231 to a final concentration of 15µM selumetinib and 11µM pimasertib by continuous exposure to increasing concentrations over a 6-month period. MEKi-resistant cells showed a significantly higher proliferation rate than the parental cells. More colonies were observed among the MEKi-resistant cells than in the parental cells in a colony formation assay (40%, p=0.05) and a similar outcome was observed in a mammosphere assay (65%, p=0.01), suggesting a higher fraction of tumor-initiating cells. To determine if the Mcl-1-specific inhibitor S63845 could sensitize the MEKi-resistant cells, we treated parental and resistant cells with either selumetinib or pimasertib together with S63845, a highly specific Mcl-1 inhibitor. After treatment with the Mcl-1 inhibitor, the resistant SUM-149 and MDA-MB-231 cells had similar cell proliferation rates to those of their parental counterparts. To extrapolate the mechanism of MEKi resistance, we performed an RT2 Profiler PCR Human Mitochondria Array; mitochondrial uncoupling proteins UCP4 and UCP2 were upregulated in the SUM149 and MDA-MB-231 MEKi-resistant cells, respectively. MDA-MB-231 MEKi-resistant cells had higher levels of glycolysis than the parental control cells, suggesting that the MEKi-resistant cells had undergone a metabolic shift to enhance glycolysis. This is consistent with a observation that metabolic alterations play a part in drug resistance. In addition, the Warbug effect, in which tumor cells adopt aerobic glycolysis, is considered the major adaptive mechanism and increased glycolysis is commonly displayed in resistant cells. Furthermore, knockdown of UCP2 in selumetinib-resistant MDA-MB-231 cells made them ~20% more sensitive to selumetinib. Inhibition of glycolysis using 2-deoxy-D-glucose in combination with MEKi increased drug sensitivity by 20-30%. Conclusion: We found preliminary evidence of a link between Mcl-1 and glycolytic metabolism that may be involved in MEK resistance. We will also design combinational studies of MEKi and Mcl-1 inhibitors in vivo in TNBC models. Citation Format: Maria Gagliardi, Moises Tacam, Lakesla Iles, Yuan Qi, Lajos Pusztai, Debu Tripathy, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Mechanism of MEK inhibitor resistance in triple negative breast cancer [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 1897.

Abstract 4841: Functional genome-wide screening identifies triple-negative breast cancer cell cycle-dependent vulnerability driven by genomic gain of death effector domain-containing protein

Abstract Triple-negative breast cancer (TNBC) persists as a clinical challenge, with targeted therapeutic options remaining limited for the subtype due to few targetable tumor-specific vulnerabilities. While basal-like TNBC tumors express epidermal growth factor receptor (EGFR), the target of multiple clinically efficacious therapies, TNBC showed little to no response to anti-EGFR therapy in clinical trials. Using a genome-wide synthetic lethal pooled barcoded shRNA screen, we identified Death Effector Domain-containing protein (DEDD), upregulated in over 60% of TNBC tumors, as a driver of mitogen-independent G1/S cell cycle transition in TNBC under EGFR inhibition. This cell cycle role of DEDD depends on cytoplasmic localization and is independent of nuclear DEDD, which maintains roles in apoptosis. Cytosolic DEDD promotes G1/S transition through direct interaction with Rb family proteins, encouraging their degradation. Further, DEDD interacts with heat shock 71kDa protein 8 (HSC70) to augment cyclin D1 expression and drive transition to S phase. These interactions of overexpressed cytosolic DEDD with the G1/S checkpoint mechanism present a vulnerability of TNBC to cell cycle inhibition. Frequent Rb-loss in TNBC has prevented clinical testing of CDK4/6 inhibitors in TNBC, however. Yet, we found TNBC with DEDD overexpression to be sensitive to a combination of CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo, independent of Rb status. This novel vulnerability presented by DEDD overexpression in TNBC suggests promising therapeutic potential for CDK4/6 inhibitor use in combinatorial regimens. Citation Format: Yingjia Ni, Keon R. Schmidt, Barnes A. Werner, Jenna K. Koenig, Ian H. Guldner, Patricia M. Schnepp, Xuejuan Tan, Lan Jiang, Misha Host, Longhua Sun, Erin N. Howe, Junmin Wu, Laurie E. Littlepage, Harikrishna Nakshatri, Siyuan Zhang. Functional genome-wide screening identifies triple-negative breast cancer cell cycle-dependent vulnerability driven by genomic gain of death effector domain-containing protein [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 4841.

Abstract 3426: A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors

Abstract Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation and minimize toxicity via MDM2i, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. By integrating the results of these two screens, we found inhibition of BRD4 activates p53 and its target genes. BRD4 (Bromodomain-containing protein 4) is a member of the bromodomain and extraterminal (BET) family proteins, which has typically been reported to activate genes, such as c-MYC, BCL2 and CDK4/6. However, we unexpectedly reveal that BRD4 acts as a transcriptional repressor of p53 target genes. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type p53 in vitro, against two mouse models of AML in vivo, and against several primary human patient blasts in vitro. Synergistic cell killing was associated with synergistic activation of p53 target genes, and cell killing and p53 target gene expression. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, targetable wild-type p53 and a transcriptional repressor function of BRD4 together represent a potential broad-spectrum synthetic therapeutic vulnerability for AML. Citation Format: Sha Li, Anne Latif, Ashley Newcombe, Kathryn Gilroy, Neil Robertson, Xue Lei, Darren Finlay, Helen Stewart, Karina Barbosa, Brian Higgins, Tim Chevassut, Xu Huang, Mhairi Copland, Karen Keeshan, Ani Deshpande, Peter Adams. A synthetic lethality approach to eradicate AML via synergistic activation of pro-apoptotic p53 by MDM2 and BET inhibitors [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 3426.