Dramatic Tumor Regression Research Articles

Epigenetic and oncogenic inhibitors cooperatively drive differentiation and kill KRAS-mutant colorectal cancers.

Current treatments for KRAS-mutant colorectal cancers (CRCs) are often limited by cellular plasticity and rewiring responses. Here we describe a promising therapeutic strategy that simultaneously targets epigenetic and oncogenic signals. Specifically, we show that inhibitors of the histone methyltransferase, EZH2, synergize with various RAS pathway inhibitors and promote dramatic tumor regression in vivo. Together these agents cooperatively suppress WNT-driven transcription and drive CRCs into a more differentiated cell state by inducing the Groucho/TLE corepressor, TLE4, along with a network of WNT pathway inhibitors and intestinal differentiation proteins. However, these agents also induce the pro-apoptotic protein BMF, which subsequently kills these more differentiated cells. Accordingly, cell death can be prevented by activating β-catenin, blocking differentiation, or by ablating BMF expression. Collectively, these studies reveal a new therapeutic approach for treating KRAS-mutant CRCs and illustrate a critical convergence of EZH2 and RAS on oncogenic WNT signals, intestinal differentiation, and apoptosis.

FLT1 activation in cancer cells promotes PARP-inhibitor resistance in breast cancer

Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer.

MALT1 Protease Inhibition Overcomes BTK Inhibitor Resistance and Shows Synergistic Activity with Venetoclax in Models of B Cell Lymphoma and Leukemia

Background Specific B cell malignancies, including the aggressive non-germinal center B cell-like (GCB) subtype of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are driven by constitutive activation of the transcription factor NF-kB. Chronic signaling resulting from mutations or antigen-induced activation of the B cell receptor (BCR) pathway drives NF-kB activity in these tumors resulting in sustained proliferation and survival pathways. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represents a key mediator of the BCR/NF-kB signal transduction pathway responsible for maintaining survival and driving proliferation of NF-kB-addicted B cell tumors by proteolytically cleaving NF-kB antagonists. Aims Pharmacological inhibition of MALT1 protease activity may provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Methods A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results ABBV-MALT1 potently inhibits MALT1 activity in vitro while showing high selectivity against a panel of proteases, kinases and other types of receptors and enzymes. Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-kB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton's tyrosine kinase (BTK) inhibitors. NF-kB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-kB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Summary/Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with DLBCL, CLL and other B cell malignancies. Disclosures All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Phase II study of niraparib in patients with advanced melanoma with genetic homologous recombination (HR) mutation/alteration.

TPS9596 Background: There are a limited number of therapeutic options available for metastatic melanoma patients progressing on the checkpoint inhibitor(s) and BRAF-targeting drugs. PARP inhibitors have been approved in the setting of BRCA1/2 germline mutations, and have demonstrated activity in the setting of HR deficiency in other cancers. Documentation of PARP inhibitors in melanoma would have clear clinical impact. We have previously shown that 21-34% of metastatic melanomas harbor at least 1 molecular aberration in the HR pathway, considered pathogenic, and likely leading to HR deficiency. Previously, we demonstrated dramatic regression of tumors harboring HR mutation(s) with PARP inhibitor treatment in a PDX model. These findings provide a strong rationale to evaluate the clinical efficacy of a PARP inhibitor in patients with advanced melanoma with HR deficiency. Methods: This is a single arm, open-label, multi-center phase II trial. The primary objective of the study is: To evaluate the clinical efficacy of niraparib in advanced melanoma patients with HR deficiency. The secondary objectives is to evaluate PFS, OS, and safety profile of niraparib. The key eligibility criteria: Advanced melanoma patients with mutation in any of the following: ARID1A/B, ARID2, ATM, ATR, ATRX, BARD1, BRCA1/2, BAP1, BRIP1, CHEK2, FANCD2, MRN11A, RAD50, RAD51, RAD54B, PALB2 (requires a genetic analysis result performed at any CLIA-certified laboratory) disease progression on PD1-antibody therapy and if BRAF mutant, BRAF/MEK inhibitors ≥ 18 years of age; ECOG PS ≤ 1; prior systemic cytotoxic therapy ≤1 regimen; no limit on number of prior immunotherapy or targeted therapy regimens. No symptomatic brain metastasis; active brain lesions ≥6 mm size, or requiring steroid treatment Patients will be treated with oral niraparib once daily (28-day cycle); the dose will be 300 mg (if weight ≥77 kg and platelet counts of ≥150,000 µL); otherwise 200 mg RECIST 1.1 will be used to evaluate clinical response every 8 weeks. Adverse events will be recorded according to CTCAE version 4.03. The primary study endpoint is overall response rate. Simon’s optimal 2-stage design is proposed. With significance level of5% and 80% power, our null hypothesis that the true response rate is 0.10 will be tested against a one-sided alternative: H0: p ≤ 10%, a response rate of no real interest VS H1: p ≥ 30%, a response rate that would be of considerable interest 10 patients will be analyzed for a response evaluation in the first stage of the study, and 19 in the second stage, for a total of 29 patients. If the response rate is 10% (1/10) or less at the end of the first stage, further accrual will be terminated. With 2 or more responses in the first stage, accrual will continue to a total of 29 patients for this cohort. The null hypothesis will be rejected if ≥6 responses are observed in a cohort of 29 subjects. Clinical trial information: NCT03925350 .

Recombinant measles virus vaccine rMV-Hu191 exerts an oncolytic effect on esophageal squamous cell carcinoma via caspase-3/GSDME-mediated pyroptosis

Oncolytic viruses have recently been proven to be an effective and promising cancer therapeutic strategy, but there is rare data about oncolytic therapy in esophageal squamous cell carcinoma (ESCC), especially oncolytic measles virotherapy. Therefore, this study aimed to explore whether the recombinant measles virus vaccine strain rMV-Hu191 has an oncolytic effect against ESCC cells in vitro and in vivo and elucidate the underlying mechanisms. Our results showed that rMV-Hu191 could efficiently replicate in and kill ESCC cells through caspase-3/GSDME-mediated pyroptosis. Mechanistically, rMV-Hu191 triggers mitochondrial dysfunction to induce pyroptosis, which is mediated by BAK (BCL2 antagonist/killer 1) or BAX (BCL2 associated X). Further analysis revealed that rMV-Hu191 activates inflammatory signaling in ESCC cells, which may enhance the oncolytic efficiency. Moreover, intratumoral injection of rMV-Hu191 induced dramatic tumor regression in an ESCC xenograft model. Collectively, these findings imply that rMV-Hu191 exhibits an antitumor effect through BAK/BAX-dependent caspase-3/GSDME-mediated pyroptosis and provides a potentially promising new therapy for ESCC treatment.

Combating castration-resistant prostate cancer by co-targeting the epigenetic regulators EZH2 and HDAC.

While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.

Abstract 6156: MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

Abstract Introduction: Specific B cell malignancies, including CLL and the aggressive non-GCB subtype of DLBCL, are driven by constitutive activation of the B cell receptor (BCR) pathway and the transcription factor NF-κB. Pharmacological inhibition of MALT1 protease, a key mediator of the BCR/NF-κB signal transduction pathway, may therefore provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Experimental Procedures: A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results: Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-κB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton’s tyrosine kinase (BTK) inhibitors. NF-κB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-κB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with B cell malignancies. Citation Format: Joshua P. Plotnik, Raghuveer S. Mali, Velitchka Bontcheva, Colleen Dowell, Jun Chen, Adam E. Richardson, Ryan A. McClure, Paul Jung, Lan Pham, Andrew J. Souers, Jonathan A. Meulbroek, William N. Pappano. MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6156.

Abstract 1781: G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma

Abstract Adoptive cell transfer (ACT) has been highly efficient in targeting certain refractory cancers and remains potentially effective for other cancers as new targets of cancer-immune interactions are revealed. ACT involves isolating immunocompetent cells from cancer patients, expanding them ex vivo, and infusing them back into the patient. Cells used for ACT are often effector cells, typically T cells, isolated from the patient’s peripheral blood and then engineered to target cancer cells by incorporating a T cell receptor (TCR) or chimeric antigen receptor (CAR), with additional modifications to improve immune cell proliferation and persistence. While cell therapies for various lymphomas have led to dramatic tumor regressions, response in solid tumors remains varied. Solid tumors present unique challenges, such as tumor heterogeneity and the lack of tumor-specific targets, but also an immunosuppressive tumor microenvironment (TME) characterized by poor T cell infiltration and terminal T cell differentiation and exhaustion at the tumor. To overcome the hostile TME, lymphodepleting regimens combined with an infusion of large numbers of T cells is often used. However, introducing large numbers of T cells significantly increases the risk of on-target/off-target toxicity, neurotoxicity, and cytokine release syndrome. The use of engineered T cells that transiently express TCRs or CARs by mRNA gene transfer, reduces these risks. Additionally, they avoid the use of viral vectors, hence there is no risk of insertional mutagenesis and can be manufactured more easily at low cost and on a larger scale. Such transient-engineered T cells have shown significant antitumor activity in phase I clinical trials and in preclinical animal models. However, multiple infusions are often required, increasing patient discomfort and treatment cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targeting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in the expression of genes and proteins involved in pro-inflammatory pathways, T cell activation, and cytotoxicity. Citation Format: Maxine S. Lam, Jose A. Reales-Calderon, Jin Rong Ow, Joey Aw, Damien Tan, Ragavi Vijayakumar, Erica Ceccarello, Tommaso Tabaglio, Yan Ting Lim, Wang Loo Chien, Fritz Lai, Anthony Tan Tanoto, Qingfeng Chen, Radoslaw M. Sobota, Giulia Adriani, Antonio Bertoletti, Ernesto Guccione, Andrea Pavesi. G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1781.

MP28-15 A NEW STRATEGY TO ENHANCE CISPLATIN EFFICACY WHILE REDUCING NEPHROTOXICITY THROUGH TARGETED NEDDYLATION INHIBITION

You have accessJournal of UrologyCME1 Apr 2023MP28-15 A NEW STRATEGY TO ENHANCE CISPLATIN EFFICACY WHILE REDUCING NEPHROTOXICITY THROUGH TARGETED NEDDYLATION INHIBITION Kyle Garcia, Trace Jones, Claudia Espitia, Juan Chipollini, Benjamin Lee, Jason Wertheim, Jennifer Carew, and Steffan Nawrocki Kyle GarciaKyle Garcia More articles by this author , Trace JonesTrace Jones More articles by this author , Claudia EspitiaClaudia Espitia More articles by this author , Juan ChipolliniJuan Chipollini More articles by this author , Benjamin LeeBenjamin Lee More articles by this author , Jason WertheimJason Wertheim More articles by this author , Jennifer CarewJennifer Carew More articles by this author , and Steffan NawrockiSteffan Nawrocki More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003256.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although cisplatin remains a backbone of standard of care chemotherapy regimens for a variety of malignancies, its use is often associated with severe dose-limiting toxicities (DLT). Notably, 30-40% of patients treated with cisplatin-based regimens are forced to discontinue treatment after experiencing nephrotoxicity as a DLT. New approaches that simultaneously prevent renal toxicity while improving therapeutic response have the potential to make a major clinical impact for patients with multiple forms of cancer. Here, we report that pevonedistat (MLN4924), a first-in-class NEDDylation inhibitor, alleviates nephrotoxicity and synergistically enhances the anticancer efficacy of cisplatin in head and neck squamous cell carcinoma (HNSCC) models METHODS: The effects of pevonedistat and cisplatin on normal renal proximal epithelial cells were evaluated using in vitro and in vivo models. Molecular and biological experiments were performed to investigate the cytoprotective effects of pevonedistat from cisplatin-mediated nephrotoxicity. RESULTS: We demonstrate that pevonedistat protects normal kidney cells from injury while enhancing the anticancer activity of cisplatin through a thioredoxin interacting protein (TXNIP)-mediated mechanism. Co-treatment with pevonedistat and cisplatin yielded dramatic HNSCC tumor regression and long-term animal survival in 100% of treated mice. Importantly, the combination decreased nephrotoxicity induced by cisplatin monotherapy as evidenced by the blockade of kidney injury molecule-1 (KIM-1) and TXNIP expression, a reduction in collapsed glomeruli and necrotic cast formation, and inhibition of cisplatin-mediated animal weight loss. CONCLUSIONS: Inhibition of NEDDylation represents a novel strategy to prevent cisplatin-induced nephrotoxicity while simultaneously enhancing its anticancer activity through a redox-mediated mechanism. A clinical trial further investigating the safety and efficacy of this approach is being planned. Source of Funding: Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award numbers T32CA009213, R01CA268383, R01CA190789 and P30CA023074 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e377 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kyle Garcia More articles by this author Trace Jones More articles by this author Claudia Espitia More articles by this author Juan Chipollini More articles by this author Benjamin Lee More articles by this author Jason Wertheim More articles by this author Jennifer Carew More articles by this author Steffan Nawrocki More articles by this author Expand All Advertisement PDF downloadLoading ...

Efficacy of pp65-specific TCR-T cell therapy in treating cytomegalovirus infection after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection remains a major cause of mortality after hematopoietic stem cell transplantation (HSCT). Current treatments, including antiviral drugs and adoptive cell therapy with CMV-specific cytotoxic T lymphocytes (CTLs), only show limited benefits in patients. T-cell receptor (TCR)-T cell therapy offers a promising option to treat CMV infections. Here, using tetramer-based screening and single-cell TCR cloning technologies, we identified various CMV antigen-specific TCRs from healthy donors, and generated TCR-T cells targeting multiple pp65 epitopes corresponding to three major HLA-A alleles. The TCR-T cells showed efficient cytotoxicity toward epitope-expressing target cells in vitro. After transfer into immune-deficient mice bearing pp65+ HLA+ tumor cells, TCR-T cells induced dramatic tumor regression and exhibited long-term persistence. In a phase I clinical trial (NCT04153279), CMV TCR-T cells were applied to treat patients with CMV reactivation after HSCT. Except one patient who withdrew at early treatment stage, all other six patients were well-tolerated and achieved complete response (CR), no more than grade 2 cytokine release syndrome (CRS) and other adverse events were observed. CMV TCR-T cells persisted up to 3months. Among them, two patients have survived for more than 1 year. This study demonstrates the great potential in the treatment and prevention of CMV infection following HSCT or other organ transplantation.

Innovative Translation Strategies in the Emergence of Next-Generation Drugs in Precision Cancer Medicine

There has been a conscientious momentum to evolve more efficacious and potent chemotherapeutics that could cure advanced cancers. Although, the conventional cancer therapeutics can cause dramatic tumor regressions in some patients, many patients inexplicably reveal no benefit. This is a unique characteristic of tumor recurrence in human cancers, linked with several resistance mechanisms. Recent past years witnessed enormous ground-breaking advancements in genomic medicine symbolizing a culmination of patients' genomic, proteomic, metabolomic, including cellular profiles which have significantly transformed understanding and therapeutic options for cancer management. Furthermore, the ability of combination chemotherapy to cure advanced cancers, immensely facilitated the prospects of adjuvant chemotherapy. Many global pharmaceuticals have accelerated translational research strategies on proteomics and genomic technologies paving the way to emerge next-generation drugs to strengthen personalized cancer medicine. Numerous forthcoming cancer therapies are at most vital phase in their innovation and development for the unfulfilled clinical need to turn into futuristic cancer treatment strategies. There is a growing optimism about the innovative strategies on emergence of the new generation of immunotherapy, targeted therapies, and Oligonucleotide therapy to improve patient outcomes, and to make real dream of accomplishing personalized cancer therapy.

Abstract ND06: ABBV-CLS-484: An active site PTPN2/N1 inhibitor that augments the immune response and sensitizes tumors to immune-mediated killing

Abstract Background: Pharmacologic inhibition of PTPN2 and PTPN1 (PTPN2/N1) represents a novel therapeutic approach in immuno-oncology that augments innate and adaptive immune responses in addition to enhancing tumor cell sensitivity to immune-mediated killing. PTPN2/N1 emerged as top hits in an in vivo CRISPR screen to identify tumor-intrinsic targets that enhance sensitivity and overcome resistance to anti-PD-1 treatment. PTPN2/N1 are phosphatases that act as negative regulators in numerous pathways including immune activation. While phosphatases have long been of interest, they are challenging drug targets, and the active site had been considered undruggable. Results: Here we report the discovery of the highly selective, active site PTPN2/N1 small molecule inhibitor, ABBV-CLS-484. Highly optimized ligand-protein interactions have led to the design of sub-nanomolar PTPN2/N1 inhibitors, confirmed through x-ray crystallography. PTPN2/N1 inhibitors increase the activation and function of cytotoxic T cells as well as increase the pro-inflammatory properties of CD103+ dendritic cells and macrophages in vitro. However, they do not cause non-specific activation in the absence of stimulation; rather, they augment signaling in cells that are already activated. PTPN2/N1 inhibition also has effects directly on tumor cells, where it amplifies sensitivity to immune-mediated killing by enhancing the interferon response. ABBV-CLS-484 promotes anti-tumor immunity as monotherapy and in combination with anti-PD-1 leading to dramatic tumor regression, even in models resistant to anti-PD-1 treatment such as 4T1, or those with minimal inflammation such as EMT6. Single-cell RNAseq analyses of tumor-infiltrating immune cells confirmed activation of T cells and demonstrated switching of myeloid-derived suppressor cells towards a proinflammatory phenotype, thereby revealing a distinct mechanism of action of ABBV-CLS-484 compared with PD-1 blockade. Our results show that PTPN2/N1 inhibitors have complementary effects on the immune system and tumor microenvironment that act to promote effective tumor killing. Based on these robust preclinical data, phase I clinical trials of ABBV-CLS-484 alone and in combination with an anti-PD-1 agent have been initiated to establish the safety, tolerability, and efficacy in diverse solid tumor indications. Conclusions: We have discovered a first-in-class PTPN2/N1 inhibitor, which represents a promising novel immunotherapy that both enhances the immune response and increases tumor sensitivity to immune-mediated killing. ABBV-CLS-484 is currently being evaluated in phase I clinical trials in patients with advanced solid tumors, as a monotherapy or in combination with a PD-1 targeting agent (NCT04777994). Citation Format: Christina K. Baumgartner, Marcia N. Paddock, Jennifer M. Frost, Keith M. Hamel, Kathleen A. McGuire, Kyle Halliwill, Zhaoming Xiong, Liang Mu, Kelly Klinge, Prasanthi Geda, Jaqueline Aguado, Marinka Bulic, Elliot P. Farney, Kathleen B. Yates, Robert T. Manguso, Clay Beauregard, Philip R. Kym. ABBV-CLS-484: An active site PTPN2/N1 inhibitor that augments the immune response and sensitizes tumors to immune-mediated killing [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 ND06.

EXTH-58. THERAPEUTIC HDAC INHIBITION IN HYPERMUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) continues to carry a dismal prognosis despite a growing understanding of its epigenetic regulation. While generally reclassified as diffuse midline glioma, H3 K27M-mutant (DMG), a subgroup of DIPGs do not harbor the classic histone mutation, with a further subset exhibiting a hypermutant phenotype. To evaluate whether hypermutant DIPG shares transcriptional vulnerabilities with H3K27M-mutant DMG, we screened a biopsy-derived treatment-naive PMS2 mutant DIPG model (PBT-24FH) for sensitivity to a panel of HDAC inhibitors (HDACi). In vitro evaluation of cell viability revealed the low nanomolar IC50 of quisinostat (50nM) and romidepsin (2nM). Dose-dependent increases in H3 acetylation and c-PARP were confirmed by western blot. Despite romidepsin’s superior potency in vitro, quisinostat demonstrated greater efficacy in an in vivo PBT-24FH flank study. 42 days following drug initiation, quisinostat-treated mice displayed dramatic tumor regression (mean volume= 33mm3, n= 7) compared to mice treated with romidepsin (mean volume= 669mm3, n= 7)(p= 0.005), or vehicle (mean volume= 990mm3, n= 6)(p< 0.001). Immunohistochemistry of quisinostat-treated tumors revealed few residual tumor cells displaying a low proliferative index. To evaluate cross-resistance, romidepsin-treated mice (mean volume= 1158mm3, n= 2) were switched to quisinostat treatment and displayed swift tumor regression (mean volume after 25 days of quisinostat= 419mm3), emphasizing quisinostat’s in vivo cytotoxic effect against both large tumors and tumors previously treated by another HDACi. To evaluate quisinostat’s effect on other hypermutant tumors, we tested HCT-116, a colon cancer cell line bearing a biallelic MLH1 deletion and observed similar cytotoxicity. We also aim to repeat these studies utilizing additional pediatric hypermutant high grade glioma models. Transcriptomic and proteomic investigations are underway to identify the mechanism of action underlying quisinostat-induced cytotoxicity. Ultimately, we are the first to demonstrate in vivo efficacy of the HDACi quisinostat against hypermutant DIPG, supporting further investigation and clinical advancement.

Subclone Eradication Analysis Identifies Targets for Enhanced Cancer Therapy and Reveals L1 Retrotransposition as a Dynamic Source of Cancer Heterogeneity.

Treatment-eradicated cancer subclones have been reported in leukemia and have recently been detected in solid tumors. Here we introduce Differential Subclone Eradication and Resistance (DSER) analysis, a method developed to identify molecular targets for improved therapy by direct comparison of genomic features of eradicated and resistant subclones in pre- and posttreatment samples from a patient with BRCA2-deficient metastatic prostate cancer. FANCI and EYA4 were identified as candidate DNA repair-related targets for converting subclones from resistant to eradicable, and RNAi-mediated depletion of FANCI confirmed it as a potential target. The EYA4 alteration was associated with adjacent L1 transposon insertion during cancer evolution upon treatment, raising questions surrounding the role of therapy in L1 activation. Both carboplatin and enzalutamide turned on L1 transposon machinery in LNCaP and VCaP but not in PC3 and 22Rv1 prostate cancer cell lines. L1 activation in LNCaP and VCaP was inhibited by the antiretroviral drug azidothymidine. L1 activation was also detected postcastration in LuCaP 77 and LuCaP 105 xenograft models and postchemotherapy in previously published time-series transcriptomic data from SCC25 head and neck cancer cells. In conclusion, DSER provides an informative intermediate step toward effective precision cancer medicine and should be tested in future studies, especially those including dramatic but temporary metastatic tumor regression. L1 transposon activation may be a modifiable source of cancer genomic heterogeneity, suggesting the potential of leveraging newly discovered triggers and blockers of L1 activity to overcome therapy resistance. SIGNIFICANCE: Differential analysis of eradicated and resistant subclones following cancer treatment identifies that L1 activity associated with resistance is induced by current therapies and blocked by the antiretroviral drug azidothymidine.

Abstract 971: Chaperone-mediated protein degradation (CHAMP): A novel technology for tumor-targeted protein degradation

Abstract The HSP90 chaperone mediates folding of many important client proteins and mutated oncoproteins, but can also direct its substrates towards degradation by the ubiquitin-proteasome system. Further, in tumor tissues, HSP90 complexes are in a highly activated state relative to normal tissues, which results in HSP90-binding small molecule compounds displaying unique tumor-selective pharmacokinetics. In order to take advantage of these attributes of HSP90 to degrade the transcription factor BRD4 in a tumor-selective fashion, hetero-bifunctional small molecule compounds were synthesized, termed chaperone-mediated protein degraders (CHAMPs), that chemically induced proximity between BRD4 and HSP90. In vitro, treatment of MV-4-11 leukemia cells with a BRD4-CHAMP compound resulted in formation of a BRD4:CHAMP:HSP90 ternary complex and subsequent proteasome-dependent BRD4 degradation and inhibition of cell proliferation. CHAMPs were identified that induced selective degradation of BRD4 and displayed only minimal effects on other HSP90-regulated client proteins. While BRD4 is a known HSP90 client protein, non-HSP90 clients such as mutated KRAS could also be degraded using this approach. In vivo, in the MV-4-11 mouse xenograft model, a selective BRD4-CHAMP compound displayed prolonged pharmacokinetics in tumors relative to plasma and normal tissues, with its tumor concentration remaining above the in vitro cytotoxicity EC50 value for at least 7 days following a single dose. This resulted in greater than 90% and prolonged degradation of BRD4 in tumors, as well as decreased expression of the BRD4-regulated MYC gene. Treatment with BRD4-CHAMP at its MTD resulted in dramatic tumor regressions after a single dose and the majority of tumors were undetectable following 3 weekly doses. In contrast, daily dosing of a clinical stage pan-BET inhibitor at its MTD resulted in stable to progressive disease, indicating that BRD4-CHAMP has a greatly improved therapeutic index in this model. CHAMP technology has a number of advantages relative to other targeted protein degradation approaches, such as PROTAC, including an improved safety margin due to selective accumulation in tumor tissues. Citation Format: Kevin P. Foley, Long Ye, Mingkai Wang, Chenghao Ying, Wei Yin, Lingjie Zhang, Weiwen Ying. Chaperone-mediated protein degradation (CHAMP): A novel technology for tumor-targeted protein degradation [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 971.

Navitoclax enhances the effectiveness of EGFR-targeted antibody-drug conjugates in PDX models of EGFR-expressing triple-negative breast cancer

BackgroundTargeted therapies for triple-negative breast cancer (TNBC) are limited; however, the epidermal growth factor receptor (EGFR) represents a potential target, as the majority of TNBC express EGFR. The purpose of these studies was to evaluate the effectiveness of two EGFR-targeted antibody-drug conjugates (ADC: ABT-414; ABBV-321) in combination with navitoclax, an antagonist of the anti-apoptotic BCL-2 and BCL-XL proteins, in order to assess the translational relevance of these combinations for TNBC.MethodsThe pre-clinical efficacy of combined treatments was evaluated in multiple patient-derived xenograft (PDX) models of TNBC. Microscopy-based dynamic BH3 profiling (DBP) was used to assess mitochondrial apoptotic signaling induced by navitoclax and/or ADC treatments, and the expression of EGFR and BCL-2/XL was analyzed in 46 triple-negative patient tumors.ResultsTreatment with navitoclax plus ABT-414 caused a significant reduction in tumor growth in five of seven PDXs and significant tumor regression in the highest EGFR-expressing PDX. Navitoclax plus ABBV-321, an EGFR-targeted ADC that displays more effective wild-type EGFR-targeting, elicited more significant tumor growth inhibition and regressions in the two highest EGFR-expressing models evaluated. The level of mitochondrial apoptotic signaling induced by single or combined drug treatments, as measured by DBP, correlated with the treatment responses observed in vivo. Lastly, the majority of triple-negative patient tumors were found to express EGFR and co-express BCL-XL and/or BCL-2.ConclusionsThe dramatic tumor regressions achieved using combined agents in pre-clinical TNBC models underscore the abilities of BCL-2/XL antagonists to enhance the effectiveness of EGFR-targeted ADCs and highlight the clinical potential for usage of such targeted ADCs to alleviate toxicities associated with combinations of BCL-2/XL inhibitors and systemic chemotherapies.

Abstract 5198: JNJ-61186372, a novel EGFR/c-Met bispecific antibody, exhibits potent antitumor activity in broad-spectrum of acquired resistance to EGFR-TKIs

Abstract Background: 1st generation (erlotinib & gefitinib), 2nd generation (afatinib), and 3rd generation (osimertinib) EGFR TKIs have marked efficacy in patient with EGFR-mutant NSCLC. Unfortunately, patients who show T790M (-) mutation after treated with 1st and 2nd generation EGFR TKI or have C797S (+) mutation or other mechanisms of acquired resistance to 3rd generation EGFR TKI have no therapeutic options for curing cancer in clinic. We introduce a potent therapeutics for overcoming acquired resistance to EGFR-TKIs in several EGFR mutant NSCLC models using JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and c-Met receptors. Methods: We classified PDC and PDX models into two groups: i) 1st and 2nd generation EGFR TKI acquired resistance models with T790M (-) mutation (YU-1093, YU-1152), ii) 3rd generation EGFR TKI acquired resistance models with C797S (+) mutation (YU-1097), and C797S (-) mutation with unknown resistance mechanisms (YU-1095, YU-1089, YU-1096, YUX-1024_YH1R, YHIM-1035, YHIM-1045, and YHIM-1053). We established 7 patient-derived cells (PDC) and 3 patient-derived xenografts (PDX) model from patients with broad-spectrum acquired resistance to EGFR-TKIs. We performed cell viability test and western blot to investigate antitumor activity of JNJ-372. We evaluated then in vivo antitumor activity of JNJ-372 in PDX and PDC-xenograft models. JNJ-372 was administrated i.p. twice a week at 30 mg/kg. Results: We assessed the ability of JNJ-372 to overcome broad-spectrum of EGFR-TKI acquired resistances. JNJ-372 significantly inhibited cancer cell growth in vitro and in vivo in PDC model harboring T790M (-) mutation. JNJ-372 significantly decreased cell proliferation rate and EGFR downstream signaling in BaF3 cell harboring Del19 (L858R)/T790M/C797S mutant and PDC model harboring Del19/T790M/C797S mutant with amplified KRAS and loss of Rb. C797S (-) cases of four PDCs and three PDX models had i) loss of T790M with c-Met amplification or other acquired resistance mutation and ii) T790M (+) with amplified c-Met. In these models we identified dramatic tumor regression and reduction of EGFR and c-Met expression in vitro and in vivo PDX and PDC-xenograft models. Interestingly, we observed combination treatment of JNJ-372 and c-Met inhibitor showed synergistic immune cell mediated cellular cytotoxicity in EGFR-TKI acquired resistance models harboring c-Met amplification. JNJ-372 exhibited potent inhibition of tumor growth in preclinical models through targeting EGFR and c-Met receptor, decreasing EGFR and c-Met receptor level, inhibiting EGFR downstream signaling cascade and activating ADCC effect in vivo. Conclusion: Our results suggest that JNJ-372 will provide potential and effective therapeutic options for patients with no therapeutic option to overcome broad-spectrum of acquired resistance to EGFR-TKIs. Citation Format: SooHwan Lee, Jiyeon Yun, Seo-Yoon Jeong, Chae Won Park, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Byoung Chul Cho. JNJ-61186372, a novel EGFR/c-Met bispecific antibody, exhibits potent antitumor activity in broad-spectrum of acquired resistance to EGFR-TKIs [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 5198.

Radiation Dose Escalation is Crucial in Anti-CTLA-4 Antibody Therapy to Enhance Local and Distant Antitumor Effect in Murine Osteosarcoma.

We previously reported that a combination of 10 Gy of X-ray irradiation and dual immune checkpoint blockade with anti-CTLA-4 (C4) and anti-PD-L1 antibodies produced a significant shrinkage of irradiated and unirradiated tumors (abscopal effect) and prolonged overall survival. However, the optimal radiation delivery regimen combined with single immune checkpoint blockade of C4 for inducing a maximum systemic antitumor response still remains unclear, particularly for patients with osteosarcoma. We used syngeneic C3H mice that were subcutaneously injected with LM8 osteosarcoma cells into both legs. C4 was administered three times, and one side of the tumor was irradiated by X-ray beams. The optimal radiation dose required to induce the abscopal effect was explored with a focus on the induction of the type-I interferon pathway. Radiation delivered in a single fraction of 10 Gy, 4.5 Gy × 3 fractions (fx), and 2 Gy × 8 fx with C4 failed to produce significant inhibition of unirradiated tumor growth compared with monotherapy with C4. Dose escalation to 16 Gy in a single fraction, or the equivalent hypofractionated dose of 8 Gy × 3 fx, which significantly increased secretion of IFN-β in vitro, produced a dramatic regression of both irradiated and unirradiated tumors and prolonged overall survival in combination with C4. Furthermore, irradiation at 16 Gy in both a single fraction and 8 Gy × 3 fx diminished regulatory T cells in the unirradiated tumor microenvironment. These results suggest that total dose escalation of radiation is crucial in C4 therapy to enhance the antitumor response in both local and distant tumors and prolonged overall survival regardless of fractionation for osteosarcoma.

Abstract P3-11-06: Pre-clinical assessment of combined ABT-263/Navitoclax and ABT-414 or ABBV-321 treatment for EGFR-expressing TNBC

Abstract Despite a notable incidence of EGFR1 copy number alterations and/or enrichment of EGFR protein in a significant fraction of TNBCs, clinical application of classical EGFR-targeted therapeutics has been discouraging. Tumor specific EGFR-targeted antibodies (ABT-806) and their antibody-drug conjugates (ADC:414;321), which eliminate side effects associated with systemic anti-EGFR treatments, represent promising alternative therapeutic approaches. 414, comprised of 806 conjugated to the powerful cytotoxic MMAF, has demonstrated notable effectiveness within EGFR1-amplified/mutated tumors. However, since TNBCs are often enriched for EGFR expression in the absence of EGFR1 amplification or mutation, we explored whether neutralization of BCL-2/XL via ABT-263/Navitoclax would enhance the effectiveness of 414. Here, we evaluated 414+263 in a panel of seven EGFR-expressing patient-derived xenograft (PDX) models of TNBC. Tumor-bearing mice were randomized into one of two groups, either 414+263 or placebos. Tumor volumes were calculated via caliper-based measurements pre- and post-treatment. 14 days post-treatment, tumor growth inhibition was observed in five out of seven combination-treated tumor models; however, consistent tumor regressions were only observed in one of these models (HCI-010). Compared to the other PDX models, HCI-010 tumors were distinguished by EGFR1 low polysomy and the highest EGFR expression levels. To further explore combined treatment within HCI-010, we evaluated single-agents. To determine EGFR relevance, we also included a non-tumor targeted ADC (095-MMAF) as a single agent or in combination with 263. Tumor growth inhibition & regressions were observed in either 263 or 414+263 treated tumors. These responses were most significant under combined treatment conditions (avg. regression=40%). Tumor growth was unaffected by 414 or 095-MMAF single agents. Tumors treated with 095-MMAF+263 were comparable to single agent 263. Based upon these results, we considered an alternative EGFR-targeted ADC (321). 321, comprised of an affinity-matured version of 806 conjugated to the powerful cytotoxic PBD, exhibits enhanced EGFR affinities and has demonstrated notable effectiveness within EGFR-overexpressing tumors. To evaluate 321 combined treatment within HCI-010, tumor-bearing mice were randomized into six groups: placebos; 263; 321; 263+321; 095-PBD; 263+095-PBD. Tumor growth inhibition & tumor regressions were maintained under 263 and, unlike 414, also observed under 321 treatments. 321 resulted in dramatic tumor regressions (avg. regression=66%). Notably, 263 enhanced the effectiveness of 321 as evidenced by even more dramatic and near complete tumor regressions (avg. regression=88%). We extended these studies to include HCI-025, an additional PDX characterized by EGFR1 low polysomy and EGFR expression levels comparable to HCI-010. To evaluate 321 combined treatment within HCI-025; we performed a similar six-group study. HCI-025 tumors were also sensitive to single agent 321 (avg. regression=36%). Similar to HCI-010 and as evidenced by dramatic tumor regressions, 263 also enhanced the effectiveness of 321 within HCI-025 (avg. regression=68%). 095-PBD and 263+095-PBD also resulted in HCI-010 and HCI-025 tumor growth inhibition & regressions; however supportive of EGFR-mediated effects, 321 responses were greater than 095-PBD and 263+321 responses were greater than 263+095-PBD. These results underscore the significant potential of BCL-2/XL-inhibitors to enhance the effectiveness of cytotoxic agents delivered via ADCs. Notably, this strategy avoids the toxicities associated with systemic chemotherapy and BCL-2/XL-inhibitors. These results also highlight the translational relevance of 321+263, within the context of EGFR-expressing TNBC. Citation Format: Jason J Zoeller, Aleksandr Vagodny, Veerle W. Daniels, Krishan Taneja, Benjamin Y. Tan, Yoko S. DeRose, Maihi Fujita, Alana L. Welm, Anthony Letai, Joel D. Leverson, Vincent Blot, Roderick T. Bronson, Deborah A. Dillon, Joan S. Brugge. Pre-clinical assessment of combined ABT-263/Navitoclax and ABT-414 or ABBV-321 treatment for EGFR-expressing TNBC [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-06.

Abstract C30: Myc amplification is a negative biomarker and a resistance mechanism to trametinib/HCQ treatment, but can be overcome by combined palbociclib/HCQ treatment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant disease responsible for ~43,000 deaths in the USA in 2017. Despite an advanced understanding of the genetics, biochemistry, and biology of pancreatic cancer, there is no effective pathway-targeted therapy for PDAC such that the standard-of-care treatment for most patients remains conventional cytotoxic chemotherapy. Although the clinical picture remains grim, progress has been made in understanding how alterations in tumor suppressors and proto-oncogenes contribute to PDAC initiation and progression. Whereas initiating mutations in KRAS promote the growth of premalignant pancreatic intraepithelial neoplasia, progression to cancer malignancy requires cooperating alterations of tumor suppressors such as TP53, CDKN2A, and/or SMAD4. Downstream of KRAS oncoproteins, the RAF®MEK®ERK MAP kinase signaling pathway plays a central role in the genesis of PDAC. However, to date, pharmacologic inhibition of this pathway has demonstrated little clinical benefit in PDAC patients. We have previously shown that combined inhibition of MEK1/2 and autophagy with chloroquine/hydroxychloroquine (CQ/HCQ) displays synergistic antiproliferative effects against PDAC cell lines in vitro, in xenografted patient-derived PDAC tumors, and treatment of a pancreatic cancer patient with trametinib/HCQ resulted in dramatic tumor regression. Unfortunately, this patient did go on to progress on trametinib/HCQ therapy and a second patient who was treated with the combination of trametinib and hydroxychloroquine was refractory to treatment. Strikingly, the only genetic difference between these two patients’ tumors at the start of trametinib/HCQ treatment was an MYC amplification, and our first patient demonstrated an acquired MYC amplification upon development of resistance. Here we demonstrate in preclinical models that overexpression of MYC in Mia-PaCa2 and PDX220 cells lines resulted in both refractory and resistant tumors. We have identified that the CDK4/6 inhibitor, palbociclib, also induces protective autophagy, and treating xenografted Mia-PaCa2 tumors overexpressing MYC with palbociclib/CQ resulted in striking tumor regression. We therefore tested the combination of palbociclib/CQ on PDX derived from our first patient upon resistance to trametinib/HCQ, which demonstrated regression and suppression of tumors when treated with palbociclib/CQ, but not palbociclib alone or trametinib/CQ. Finally, we treated our first patient with the combination of palbociclib/HCQ and saw a dramatic decline in CA19-9. These data suggest that MYC amplification may be a negative biomarker for response and also a resistance mechanism that can be overcome with combined inhibition of CDK4/6 and autophagy. Citation Format: Conan Kinsey, Dilru Silva, Mark Silvis, Sophia Schuman, Kajsa Affolter, Martin McMahon. Myc amplification is a negative biomarker and a resistance mechanism to trametinib/HCQ treatment, but can be overcome by combined palbociclib/HCQ treatment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C30.