Inhibitors In Preclinical Models Research Articles

Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription.

MYC+ lymphomas are refractory to standard of care and novel treatments that downregulate MYC are needed. The utility of enitociclib, a selective CDK9 inhibitor in this patient population, is demonstrated in preclinical models and patients. Enitociclib inhibits RNA polymerase II function conferring a transcriptional shift and depletion of MYC and MCL1. Enitociclib intermittent dosing downregulates transcription factors including MYC, providing a therapeutic window for durable responses in patients with MYC+ lymphoma.

SAT071 Inhibition Of 12 Lipoxygenase In A Humanized Mouse Model Of Type 1 Diabetes Delays Progression Of Hyperglycemia

Abstract Disclosure: T. Nargis: None. A. Chakraborty: None. K. Figatner: None. D. Maloney: None. M. Boxer: None. S.A. Tersey: None. R.G. Mirmira: None. Type 1 diabetes (T1D) is an autoimmune disorder characterized by islet inflammation (insulitis). We have recently shown that a key mediator of inflammatory signaling in the islet beta cell is the enzyme 12-lipoxygenase (12-LOX), which generates proinflammatory eicosanoids that augment dysfunctional insulin secretion and beta-cell visibility to the immune system. Whereas inhibition of 12-LOX offers an opportunity to modify T1D progression by altering how the beta cell responds to insulitis, a barrier to testing inhibitors in preclinical models is the specificity of next generation inhibitors toward the human enzyme. To generate a preclinical platform for the study of human-specific 12-LOX inhibitors, we developed a mouse model in which the mouse-equivalent Alox15 coding region was replaced with the human ALOX12 gene, while retaining the mouse upstream control region. These mice (hALOX12) were developed on both the C57BL/6J and NOD genetic backgrounds, then subjected to treatment with the human 12-LOX inhibitor VLX-1005 to evaluate diabetes progression. C57BL/6J-wildtype and C57BL/6J-hALOX12 mice were confirmed to express exclusively mouse Alox15 and human ALOX12, respectively, using RNA isolated from islets. To test susceptibility to toxin-induced diabetes, C57BL/6J-wildtype and C57BL/6J-hALOX12 were subjected to multiple low-dose streptozotocin (STZ) to induce diabetes. Both wildtype and humanized mice exhibited gradual and equivalent hyperglycemia (within 25 days) following STZ injection. Following intraperitoneal VLX-1005 therapy (30 mg/kg), whereas C57BL/6J-wildtype mice developed diabetes (with a delay of 10 days) following STZ, C57BL/6J-hALOX12 mice showed complete protection against STZ-induced diabetes and exhibited significantly better glucose tolerance compared to vehicle- or VLX-1005-injected wildtype controls and vehicle-injected C57BL/6J-hALOX12 mice. Next, hALOX12 mice were backcrossed onto the NOD.ShiltJ background model of autoimmune T1D. NOD-hALOX12 mice received either vehicle or VLX-1005 (30 mg/kg) orally during the prediabetic phase (6-10 weeks of age) and were followed for diabetes incidence. Mice receiving VLX-1005 showed a significant delay with only 30% of female and 7% of male mice developing diabetes over 25 weeks of age. Pancreatic sections from the mice treated with VLX-1005 showed reduced insulitis and increased beta cell mass compared to vehicle controls. In summary, the hALOX12 mouse model serves as a preclinical translational platform to interrogate effects of next generation 12-LOX inhibitors and demonstrates the potential of VLX-1005 to modify T1D disease progression. Presentation: Saturday, June 17, 2023

Anticancer Efficacy of KRASG12C Inhibitors Is Potentiated by PAK4 Inhibitor KPT9274 in Preclinical Models of KRASG12C-Mutant Pancreatic and Lung Cancers.

KRASG12C inhibitors, such as sotorasib and adagrasib, have revolutionized cancer treatment for patients with KRASG12C-mutant tumors. However, patients receiving these agents as monotherapy often develop drug resistance. To address this issue, we evaluated the combination of the PAK4 inhibitor KPT9274 and KRASG12C inhibitors in preclinical models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). PAK4 is a hub molecule that links several major signaling pathways and is known for its tumorigenic role in mutant Ras-driven cancers. We found that cancer cells resistant to KRASG12C inhibitor were sensitive to KPT9274-induced growth inhibition. Furthermore, KPT9274 synergized with sotorasib and adagrasib to inhibit the growth of KRASG12C-mutant cancer cells and reduce their clonogenic potential. Mechanistically, this combination suppressed cell growth signaling and downregulated cell-cycle markers. In a PDAC cell line-derived xenograft (CDX) model, the combination of a suboptimal dose of KPT9274 with sotorasib significantly reduced the tumor burden (P= 0.002). Similarly, potent antitumor efficacy was observed in an NSCLC CDX model, in which KPT9274, given as maintenance therapy, prevented tumor relapse following the discontinuation of sotorasib treatment (P= 0.0001). Moreover, the combination of KPT9274 and sotorasib enhances survival. In conclusion, this is the first study to demonstrate that KRASG12C inhibitors can synergize with the PAK4 inhibitor KPT9274 and combining KRASG12C inhibitors with KPT9274 can lead to remarkably enhanced antitumor activity and survival benefits, providing a novel combination therapy for patients with cancer who do not respond or develop resistance to KRASG12C inhibitor treatment.

BRD4 as a Therapeutic Target in Pulmonary Diseases.

Bromodomain and extra-terminal domain (BET) proteins are epigenetic modulators that regulate gene transcription through interacting with acetylated lysine residues of histone proteins. BET proteins have multiple roles in regulating key cellular functions such as cell proliferation, differentiation, inflammation, oxidative and redox balance, and immune responses. As a result, BET proteins have been found to be actively involved in a broad range of human lung diseases including acute lung inflammation, asthma, pulmonary arterial hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD). Due to the identification of specific small molecular inhibitors of BET proteins, targeting BET in these lung diseases has become an area of increasing interest. Emerging evidence has demonstrated the beneficial effects of BET inhibitors in preclinical models of various human lung diseases. This is, in general, largely related to the ability of BET proteins to bind to promoters of genes that are critical for inflammation, differentiation, and beyond. By modulating these critical genes, BET proteins are integrated into the pathogenesis of disease progression. The intrinsic histone acetyltransferase activity of bromodomain-containing protein 4 (BRD4) is of particular interest, seems to act independently of its bromodomain binding activity, and has implication in some contexts. In this review, we provide a brief overview of the research on BET proteins with a focus on BRD4 in several major human lung diseases, the underlying molecular mechanisms, as well as findings of targeting BET proteins using pharmaceutical inhibitors in different lung diseases preclinically.

LRRK2 Kinase Inhibition Attenuates Neuroinflammation and Cytotoxicity in Animal Models of Alzheimer's and Parkinson's Disease-Related Neuroinflammation.

Chronic neuroinflammation plays a crucial role in the progression of several neurodegenerative diseases (NDDs), including Parkinson's disease (PD) and Alzheimer's disease (AD). Intriguingly, in the last decade, leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic PD, was revealed as a key mediator of neuroinflammation. Therefore, the anti-inflammatory properties of LRRK2 inhibitors have started to be considered as a disease-modifying treatment for PD; however, to date, there is little evidence on the beneficial effects of targeting LRRK2-related neuroinflammation in preclinical models. In this study, we further validated LRRK2 kinase modulation as a pharmacological intervention in preclinical models of AD- and PD-related neuroinflammation. Specifically, we reported that LRRK2 kinase inhibition with MLi2 and PF-06447475 (PF) molecules attenuated neuroinflammation, gliosis and cytotoxicity in mice with intracerebral injection of Aβ1-42 fibrils or α-syn preformed fibrils (pffs). Moreover, for the first time in vivo, we showed that LRRK2 kinase activity participates in AD-related neuroinflammation and therefore might contribute to AD pathogenesis. Overall, our findings added evidence on the anti-inflammatory effects of LRRK2 kinase inhibition in preclinical models and indicate that targeting LRRK2 activity could be a disease-modifying treatment for NDDs with an inflammatory component.

Vincristine Enhances the Efficacy of MEK Inhibitors in Preclinical Models of KRAS-mutant Colorectal Cancer.

Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.

CDK4/6 and autophagy inhibitors synergize to suppress the growth of human head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) accounts for over 10,000 deaths in the United States annually. Approximately 80% of HNSCC are human papillomavirus (HPV)-negative which have an overall poorer prognosis compared to the HPV-positive disease. Treatment options are mainly nontargeted chemotherapy, radiation, and surgery. The cyclin-d-CDK4/6-RB pathway, which regulates cell cycle progression, is often deregulated in HNSCC, making it an attractive therapeutic target. In the current study, we investigated the therapeutic effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors in preclinical models of HNSCCs. Our results show that the specific CDK4/6 inhibitor, abemaciclib, inhibited cell growth, and induced apoptosis in HNSCC cell lines. We also demonstrated that both the pro-survival autophagy pathway and the ERK pathway in HNSCC cells were activated with abemaciclib treatment through the generation of reactive oxygen species (ROS). Coinhibition of CDK4/6 and autophagy synergistically decreased cell viability, induced apoptosis, and inhibited tumor growth in both in vitro and in vivo preclinical HNSCC models. These results reveal a potential therapeutic strategy that supports the rationale for further clinical development of a combination of CDK4/6 and autophagy inhibitors in HNSCC.

Abstract A43: Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML

Abstract Background: RAS/RAF/ERK pathway activation is associated with primary or secondary resistance to venetoclax in AML. Oncogenic RAS activates mitochondrial fission through ERK1/2 mediated phosphorylation of the mitochondrial fission GTPase Drp1. Although the deletion of Drp1 inhibits KRAS driven pancreatic cancer, the role of RAS-driven mitochondrial dynamics in AML is unclear. Inhibiting ERK1/2 may, therefore, shed light on AML cells' dependence on RAS driven mitochondrial dynamics and metabolism. Objectives: We assessed the anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and further investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., 2018), sensitized intrinsically resistant and NRAS-mutant OCI-AML3 cells to venetoclax with combination index (CI) of 0.008. Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax (75% apoptosis versus 22% by venetoclax, CI=0.6). Bulk and CD34+CD38- leukemia initiating cells (LICs) were also eliminated in primary AML samples by the combination (CI:0.03-0.23). Colony formation assay and CyTOF using NRAS-mutant PDX showed impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to ERK1/2 inhibition and combination treatment, suggesting impact on stemness. ERK1/2 inhibition using ASTX029 (Heightman et al., 2021) alone and in combination with venetoclax reduced leukemia burden in-vivo and extended survival (p<0.05). Mitochondrial ROS mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML. ERK1/2 inhibition decreased pDrp1-Ser616 levels along with an increase in mitochondrial length (p<0.001) and mitochondrial ROS (p<0.01), suggesting impaired mitochondrial fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Overexpression of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells decreased apoptosis and reversed synergy between ERK1/2 and Bcl-2 inhibition as compared to wild type and phospho-null (Ser616Ala) Drp1 (p<0.001). Of note, overexpression of Drp1-phospho-mimetic led to decreased mitochondrial length, suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, significantly decreased maximal oxygen consumption rate, ATP production and ROS production. Moreover, combination treatment failed to significantly induce ROS production in cells expressing phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Conclusion: Inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. In summary, these data support combining ERK1/2 and Bcl-2 inhibitors in the treatment of AML and warrant further clinical investigation. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Anna Zal, Christopher Hindley, Kim-Hien Dao, Martin Sims, Tomasz Zal, Vivian Ruvolo, Michael Andreeff, Gautam Borthakur. Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A43.

Abstract LB203: Targeted inhibition of IRAK-4 with CA-4948 (emavusertib) sensitizes metastatic brain melanoma to anti-PD-1 immune checkpoint blockade

Abstract Introduction: The unique microenvironment within the brain alongside restrictions imparted by the blood brain barrier (BBB) create a sanctuary site for tumors to thrive. Melanoma is often responsive to therapy when present in peripheral tissue, but becomes recalcitrant when sequestered beyond the BBB. An estimated 40-60% of metastatic melanoma patients will develop a brain lesion, even while receiving treatment. Stereotactic radiosurgery (SRS) is the gold standard in intervention for metastatic brain melanoma (MBM), often given with anti-PD1 immune checkpoint blockade (ICB). While this therapy is initially effective, resistance occurs rapidly and ~50% of patients will develop new brain lesions within a year. Treatments at this stage are limited, necessitating the discovery of new therapies. Recent studies have shown that myeloid differentiation primary response protein 88 (MyD88) is activated in melanoma in tumor-associated myeloid cells where chronic myddosome signaling results in defective immunological responses. We have found that myddosome signaling is also prevalent in MBM, likely protecting brain metastases against effector immune detection. We recently identified that CA-4948, a small molecule inhibitor of interleukin (IL)-1 receptor-associated kinase (IRAK-4) and rate-limiting step in the myddosome cascade, can reach therapeutic levels in the brain and demonstrates on-target inhibition in preclinical models of MBM. These data highlight a unique therapeutic opportunity where selective inhibition of pro-tumorigenic cells in the MBM tumor microenvironment (TME) via targeted IRAK-4 inhibition may recondition the immunological landscape of these tumors, sensitizing these difficult-to-treat tumors to immunotherapy. Hypothesis: Selective inhibition of pro-tumorigenic cells via targeted inhibition of IRAK-4 with CA-4948 will recondition the immunological landscape of MBM, improving anti-tumor response to anti-PD-1 ICB. Methods: Patient MBM samples were assessed by IHC to define the distribution of myddosome signaling. Preclinical syngeneic MBM tumor models were treated with CA-4948 alone and in combination with anti-PD-1 ICB. Tumors were examined by flow cytometry and IHC to characterize the composition of the immune TME and evaluate the distribution of T lymphocytes. Overall survival was compared between control, single agent, and combination treated animals. Results: Tumor-associated immune cells and astrocytes comprise the majority of active myddosome signaling in patient MBM. Preclinical models corroborate these findings, with targeted inhibition of IRAK-4 through CA-4948 treatment effective at reducing activation and validating anti-tumor activity in the CNS. Analysis of treated tumors reveal decreased expression of Programmed Death Ligand 1 (PD-L1), and increased numbers of tumor-infiltrating T lymphocytes. Combination CA-4948 with anti-PD-1 ICB resulted in reduced tumor growth, and improved survival over single agent therapy. Conclusions: Therapeutic inhibition of IRAK-4 in MBM mitigates immune suppression mediated by tumor-associated cells, allowing for improved immune surveillance and tumor infiltration by effector T lymphocytes, resulting in enhanced anti-tumor activity. Thus, CA-4948 may be an effective treatment alongside anti-PD-1 ICB for patients with MBM. Citation Format: Christina A. von Roemeling, Bently P. Doonan, Rylynn A. Russell, Tyler V. Elliott, Erica Matich, Vincent Archibald, Lan Hoang-Minh, Reinhard W. von Roemeling, Dora Ferrari, Jamison Hoffman, Duane A. Mitchell. Targeted inhibition of IRAK-4 with CA-4948 (emavusertib) sensitizes metastatic brain melanoma to anti-PD-1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB203.

Abstract CT029: Phase Ib study of GDC-6036 in combination with cetuximab in patients with colorectal cancer (CRC) with KRAS G12C mutation

Abstract Background: GDC-6036 is an oral, highly potent and selective KRAS G12C inhibitor that demonstrated anti-tumor activity in patients with KRAS G12C-positive advanced solid tumors, including CRC. In a previously reported single-agent cohort, GDC-6036 achieved a best response of partial response or complete response in 35% (19/55) of patients, with a confirmed overall response rate (ORR) of 24% (13/55 patients) in KRAS G12C-positive CRC patients (Desai et. al., ESMO 2022). EGFR blockade may sensitize tumors to KRAS G12C inhibition and the combination of an anti-EGFR antibody (cetuximab) with a KRAS G12C inhibitor showed greater anti-tumor activity than single-agent KRAS G12C inhibition in preclinical models (Amodio et. al., 2020). Methods: In an ongoing Phase I study (NCT04449874), patients with advanced or metastatic KRAS G12C-positive CRC were administered GDC-6036 (200-400 mg orally once a day) with cetuximab (400 mg/m2 intravenously initially, then 250 mg/m2 weekly) until intolerable toxicity or disease progression. Endpoints included safety (NCI-CTCAE v5), pharmacokinetics (PK), and preliminary anti-tumor activity (RECIST v1.1). Results: As of the clinical data cut-off date of 21 Nov 2022, 29 patients (enrolled by 07 Oct 2022) had received GDC-6036 and cetuximab. The median lines of prior metastatic therapy was 2 (range 1-8) and the median time on study treatment was 5.2 (range 1.4-11.2) months. All patients experienced at least one treatment-related adverse event (TRAE); the most common TRAEs (≥15%) were rash (grouped terms), diarrhea, nausea, vomiting, dry skin, and paronychia. Grade 3-4 TRAEs occurred in 11 patients (38%). Five patients (17%) experienced at least one serious AE, none of which were treatment-related (including 2 patients who died of CRC progression during the safety follow-up). AEs led to GDC-6036 modifications (interruptions and/or reductions) in 13 (45%) patients, dose reduction in 3 (10%) patients, and no patients discontinued due to AEs. Eleven patients discontinued from study treatment (10 due to disease progression and 1 due to physician’s discretion). The PK profile of GDC-6036 (400 mg once a day) was similar in combination with cetuximab when compared with single-agent. A partial response was achieved in 66% (19/29) of patients, with a confirmed ORR of 62% (18/29 patients). Conclusions: GDC-6036 in combination with cetuximab demonstrated a manageable safety profile and promising clinical activity. These data support that the addition of anti-EGFR therapy to GDC-6036 may lead to robust clinical benefit in patients with KRAS G12C-positive CRC. Citation Format: Jayesh Desai, Sae-Won Han, Jong-Seok Lee, Einat Shacham-Shmueli, Erminia Massarelli, Andrés Cervantes, Elena Garralda, Alejandro Falcon, Wilson H. Miller, Eelke Gort, Thomas Karasic, Salvatore Siena, Rafal Stec, Laura Medina, Luis Paz-Arez, Angelo Delmonte, Adrian Sacher, Hans Prenen, Martin Forster, Tae Won Kim, Matthew G. Krebs, Rasha Cosman, Yoonha Choi, Sandhya Mandlekar, Mark T. Lin, Kenneth K. Yau, Julie Chang, Stephanie Royer-Joo, Neekesh V. Dharia, Jennifer L. Schutzman, Manish Patel. Phase Ib study of GDC-6036 in combination with cetuximab in patients with colorectal cancer (CRC) with KRAS G12C mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT029.

Abstract 6127: MDM2 inhibition in combination with MEK inhibition in pre-clinical models of lung adenocarcinomas with MDM2 amplification

Abstract The eventual development of resistance to single-agent targeted therapies in lung adenocarcinomas (LUAD) is inevitable, and new strategies are needed. We hypothesize that combination therapies aimed at a known driver and a distinct targetable alteration could prolong time on oral targeted therapy. In an analysis of 7636 patients with LUAD who underwent MSK-IMPACT large panel NGS testing, 5.5% (416/7636) harbored MDM2 amplification (MDM2amp), a known mechanism of TP53 inactivation. MDM2amp was over-represented among tumors with alterations in METex14 (34.4%, p<0.001), EGFR (10%, p<0.001), RET (11%, p<0.05), and ALK (9.9%, p<0.002). The small molecule MDM2 inhibitor milademetan (mila) caused growth inhibition as a single-agent in MDM2amp patient-derived cell lines with concurrent kinase alterations including ECLC5-GLx (MDM2amp/TRIM33::RET/TP53 wildtype (WT)) and LUAD12c (MDM2amp/METex14/KRASG12S/TP53 WT). Mila also caused growth inhibition in a cell line with KRASG12C and WT TP53 without MDM2amp (SW1573 (KRASG12C/TP53WT)), but not in cell lines with TP53 mutations (LUAD-002AS1 (KIF5B::RET/TP53P128fs, H1792 (KRASG12C/TP53 splice site mut)). Treatment of ECLC5-GLx and LUAD12c with mila resulted in restoration of ERK phosphorylation, confirming a previous report of ERK activation upon MDM2 inhibition. At 48 hours, ERK phosphorylation was suppressed by concurrent mila and MEK inhibition using trametinib (tram). In contrast, ERK phosphorylation was not suppressed by concurrent mila and KIF5B::RET inhibition using selpercatinib (in ECLC5-GLx) or MET inhibition using capmatinib (in LUAD12c). The combination of mila+tram was synergistic in slowing growth of ECLC5-GLx, LUAD12c, and SW1573 cells, and increased expression of pro-apoptotic proteins PUMA and BIM, beyond that achieved by either agent alone. In ECLC5-GLx, mila+tram also caused increased apoptotic cells measured by Annexin-V compared to either agent alone (combination p<0.01 compared to mila, p<0.001 compared to tram). In vivo, combination mila+tram was more effective than mila or tram alone in ECLC5-GLx (p<0.0001 and p<0.0001, respectively), LX-285 (EGFRex19del/MDM2amp) (p<0.0001 and p<0.0001, respectively), and L-13BS1 (model resistant to capmatinib) (METex14/MDM2amp) (p<0.05 and p<0.0001, respectively). These results suggest that combined MDM2/MEK inhibition is effective in patient-derived LUAD models harboring MDM2amp. This combination, potentially applicable to LUADs with a wide variety of oncogenic driver mutations and kinase fusions will be investigated as part of a phase 1/2 clinical trial. Citation Format: Arielle Elkrief, Vladimir Markov, Álvaro Quintanal-Villalonga, Rebecca Caeser, Pawel Sobczuk, Emily Cheng, Alexander Drilon, Gregory J. Riely, William W. Lockwood, Elisa de Stanchina, Charles M. Rudin, Igor Odintsov, Romel Somwar. MDM2 inhibition in combination with MEK inhibition in pre-clinical models of lung adenocarcinomas with MDM2 amplification [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 6127.

Abstract 1813: Selective ITK blockade induces antitumor responses and enhances efficacy to immune checkpoint inhibitors in preclinical models

Abstract Interleukin 2 inducible T cell kinase (ITK) plays a role in both T cell receptor (TCR) signaling and T helper cell differentiation. ITK-/- mice exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells that secrete IFNγ. We generated CPI-818, a covalent inhibitor of ITK (KD 2.5nM) with > 100 fold selectivity over resting lymphocyte kinase (RLK) and other TEC family kinases. This drug is now being evaluated as a single agent in an ongoing Phase 1 clinical trial in refractory T cell lymphoma where objective tumor responses have been observed. In the studies reported here, we have investigated the immunomodulatory effects of specific ITK blockade of human T cells with CPI-818 in vitro and in vivo in murine tumor models. Analysis of the cytokine profiles in human CD4 T cells differentiated under non-polarizing, Th1 or Th2 polarizing conditions showed that CPI-818 inhibited generation of Th2-associated cytokines whereas, production of Th1 cytokines such as IFNγ were largely unaffected. Suppression of IL-2-mediated proliferative response occurred only at high concentrations of CPI-818 (>1uM). The effect of CPI-818 on anti-tumor immunity was evaluated in both murine syngeneic CT26 colon cancer and EL4 T cell lymphoma models. In vivo treatment of animals with established tumors showed single-agent anti-tumor activity in both models. Combining CPI-818 with suboptimal doses of anti-PD1 and anti-CTLA4 synergistically inhibited the growth of established CT26 tumors, leading to complete tumor elimination in 100% treated animals. Furthermore, triple combination therapy elicited durable anti-tumor immune memory after animals were rechallenged with a new engraftment of CT26 cells. Enhancement of anti-tumor activity required CD8+ T cells as in vivo antibody-mediated depletion of CD8, but not CD4, abolished the efficacy of CPI-818 monotherapy. Consistent with that, we found increased tumor-infiltrating CD8+ T cells and increased ratio of CD8: Treg in the responding tumors of CPI-818 treated animals. Levels of several exhaustion makers were down-regulated by treatment with CPI-818, suggesting that inhibition of ITK by CPI-818 produces favorable changes in the tumor microenvironment. Our findings provide insights into the effects of selective ITK blockade on tumor immunity and its potential role in immunotherapy. Citation Format: Lih-Yun Hsu, Rahul D. Pawar, Dan Li, Poorva Ghosh, Kevin Nguyen, Carlene Williams, Yuqin Song, Ning Ding, Erik Verner, Richard A. Miller. Selective ITK blockade induces antitumor responses and enhances efficacy to immune checkpoint inhibitors in preclinical models [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 1813.

Abstract 5081: Anti-tumor activity of HPN217, a BCMA-targeting tri-specific T cell engager, is enhanced by γ-secretase inhibitors in preclinical models

Abstract HPN217 is a Tri-specific T Cell-Activating Construct (TriTAC) currently being evaluated in a phase 1 clinical trial for relapsed or refractory multiple myeloma (MM) (NCT04184050). It consists of a single domain antibody (sdAb) specific for B-cell maturation antigen (BCMA), a serum albumin-specific sdAb for half-life extension, and a single chain Fv (scFv) specific for the CD3ε subunit of the T cell receptor (TCR) complex for T cell engagement. BCMA is a cell surface receptor highly expressed on malignant plasma cells in MM patients and plays an important role in B-cell proliferation and survival. Soluble BCMA (sBCMA) is produced when the extracellular domain of BCMA is cleaved by γ-secretase and enters circulation. sBCMA may act as a sink for BCMA targeted therapies, potentially decreasing anti-tumor efficacy. Previous studies have shown γ-secretase inhibitors (GSIs) can increase membrane bound BCMA expression on MM cells and decrease sBCMA concentrations, providing a rationale for combining GSIs with HPN217. Here we describe preclinical studies evaluating that combination in vitro and in vivo. To evaluate the effects of GSIs on BCMA expression, MM cell lines MOLP8, KMS-12-BM, and MM.1S were incubated with GSIs LY3039478, PF03084014, or RO4929097overnight. GSI treatment led to a 3-7-fold increase in BCMA expression on MM cell surface. To determine the effects of increased BCMA expression mediated by GSIs on the activity of HPN217, an in vitro T cell dependent cell cytotoxicity assay (TDCC) was conducted. GSIs enhanced the activity of HPN217, in some cases leading to a 3.5-fold increase in potency compared to HPN217 alone. Next, the impact of the combination was tested in vivo using a disseminated MOLP-8 xenograft model engrafted with T cells from a healthy human donor. Vehicle treated mice succumbed to disease burden with a median survival of 23 days as assessed by body weight loss and clinical observations. A suboptimal dose of 4 ug/kg HPN217 protected mice from disease burden for an additional 3 days. By contrast, the combination of HPN217 and LY3039478 led to a significant increase in survival compared to vehicle or either monotherapy, with a median survival of 43 days. Taken together these results demonstrate the potential utility of GSIs to enhance the anti-tumor activity of HPN217 and supports further investigation of this combinatorial approach in patients. Citation Format: Payton C. Laurie, Mary Ellen Molloy, Patrick P. Ng, Banmeet S. Anand. Anti-tumor activity of HPN217, a BCMA-targeting tri-specific T cell engager, is enhanced by γ-secretase inhibitors in preclinical models. [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 5081.

Abstract 3075: Enhanced efficacy of ARV-471, a novel PROTAC® estrogen receptor degrader, in combination with targeted agents in estrogen receptor-positive (ER+) breast cancer models

Abstract ARV-471 is a selective, orally bioavailable PROteolysis-TArgeting Chimera (PROTAC®) small molecule that induces wild-type and mutant estrogen receptor (ER) alpha degradation via the ubiquitin-proteasome system. ARV-471 demonstrates superior ER degradation and antitumor activity compared to fulvestrant in endocrine sensitive and resistant xenograft models and has shown significant ER degradation and promising clinical benefit in late-line ER+ breast cancer patients. Dual pathway inhibition combining ER targeting agents with CDK4/6 or PIK3CA/mTOR pathway inhibitors is now a central strategy for treatment of advanced ER+ breast cancer. However, resistance to aromatase inhibitors resulting from ESR1 gene mutations, suboptimal ER degradation and the intramuscular route of administration of fulvestrant underscore a need for superior orally bioavailable endocrine therapy backbones for these combinations. Here, we assessed the effects of ARV-471 in combination with CDK4/6 or PIK3CA/mTOR pathway inhibitors in preclinical models of ER+ breast cancer. In vitro studies revealed evidence of synergistic interactions between ARV-471 and the CDK4/6 inhibitors abemaciclib and ribociclib, the mTOR inhibitor everolimus, or the PIK3CA inhibitors alpelisib and inavolisib in ER+ breast cancer cells. Evidence of synergistic effects between ARV-471 and everolimus was also observed in ER+ breast cancer cells expressing ER Y537S or D538G mutations. ARV-471 in combination with CDK4/6, PIK3CA or mTOR inhibitors led to tumor regressions in MCF7 xenografts when compared to single agents alone. ARV-471 also displayed greater anti-tumor activity in combination with abemaciclib, ribociclib or inavolisib than that observed with fulvestrant in combination with these agents. Taken together, these data suggest the potential utility of ARV-471 as a combination partner for clinically relevant targeted agents for treatment of early and late-stage ER+ disease. Citation Format: Jessica Teh, Elizabeth Bortolon, Jennifer Pizzano, Melissa Pannone, Sean Landrette, Richard Gedrich, Ian Taylor. Enhanced efficacy of ARV-471, a novel PROTAC® estrogen receptor degrader, in combination with targeted agents in estrogen receptor-positive (ER+) breast cancer models [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 3075.

Cav 1.3-selective inhibitors of voltage-gated L-type Ca2+ channels: Fact or (still) fiction?

Voltage-gated L-type Ca2+ -channels (LTCCs) are the target of Ca2+ -channel blockers (CCBs), which are in clinical use for the evidence-based treatment of hypertension and angina. Their cardiovascular effects are largely mediated by the Cav 1.2-subtype. However, based on our current understanding of their physiological and pathophysiological roles, Cav 1.3 LTCCs also appear as attractive drug targets for the therapy of various diseases, including treatment-resistant hypertension, spasticity after spinal cord injury and neuroprotection in Parkinson's disease. Since CCBs inhibit both Cav 1.2 and Cav 1.3, Cav 1.3-selective inhibitors would be valuable tools to validate the therapeutic potential of Cav 1.3 channel inhibition in preclinical models. Despite a number of publications reporting the discovery of Cav 1.3-selective blockers, their selectivity remains controversial. We conclude that at present no pharmacological tools exist that are suitable to confirm or refute a role of Cav 1.3 channels in cellular responses. We also suggest essential criteria for a small molecule to be considered Cav 1.3-selective.

Low-Intensity Pulsed Ultrasound-Mediated Blood-Brain Barrier Opening Increases Anti-Programmed Death-Ligand 1 Delivery and Efficacy in Gl261 Mouse Model

Therapeutic antibodies targeting immune checkpoints have shown limited efficacy in clinical trials in glioblastoma (GBM) patients. Ultrasound-mediated blood-brain barrier opening (UMBO) using low-intensity pulsed ultrasound improved drug delivery to the brain. We explored the safety and the efficacy of UMBO plus immune checkpoint inhibitors in preclinical models of GBM. A blood-brain barrier (BBB) opening was performed using a 1 MHz preclinical ultrasound system in combination with 10 µL/g microbubbles. Brain penetration of immune checkpoint inhibitors was determined, and immune cell populations were evaluated using flow cytometry. The impact of repeated treatments on survival was determined. In syngeneic GL261-bearing immunocompetent mice, we showed that UMBO safely and repeatedly opened the BBB. BBB opening was confirmed visually and microscopically using Evans blue dye and magnetic resonance imaging. UMBO plus anti-PDL-1 was associated with a significant improvement of overall survival compared to anti-PD-L1 alone. Using mass spectroscopy, we showed that the penetration of therapeutic antibodies can be increased when delivered intravenously compared to non-sonicated brains. Furthermore, we observed an enhancement of activated microglia percentage when combined with anti-PD-L1. Here, we report that the combination of UMBO and anti-PD-L1 dramatically increases GL261-bearing mice's survival compared to their counterparts treated with anti-PD-L1 alone. Our study highlights the BBB as a limitation to overcome in order to increase the efficacy of anti-PD-L1 in GBM and supports clinical trials combining UMBO and in GBM patients.

Phase I Study of Ixazomib with Conventional Chemotherapy in the Treatment of Acute Myeloid Leukemia in Older Adults

Introduction: Outcome in acute myeloid leukemia (AML) for patients aged >60 years is poor with a median survival of approximately 11 months. Targeting the proteasome in AML is attractive, since leukemia stem cells demonstrate sensitivity to proteasome inhibition in preclinical models, perhaps through down regulation of nuclear NF-KB or MCL-1 through stabilizing effects on NOXA. AML cell lines are susceptible to synergistic cytotoxicity when bortezomib, a proteasome inhibitor, is combined with daunorubicin and cytarabine. We have shown that adding bortezomib to standard treatment in AML results in a high remission rate. A newer generation proteasome inhibitor, ixazomib, is less frequently associated with neurotoxicity, and, therefore, was selected for combination with conventional chemotherapy in this phase I trial. The primary objective of this study was to determine the recommended phase II dose (RP2D) of ixazomib in combination with intensive induction and consolidation chemotherapy for AML. A secondary objective was to determine if the level of CD74 in blasts correlated with response, since prior studies showed that CD74 may be a poor prognostic indicator, but its effect might be diminished with proteasome inhibition. Methods: Adults >60 years of age with newly diagnosed AML were screened for eligibility. Patients with secondary AML were eligible, including those with prior hypomethylating agent (HMA) therapy for myelodysplastic syndromes (MDS). We excluded those with promyelocytic leukemia. There were 2 phases in this study. In the first phase (A), the induction treatment consisted of the following: cytarabine 100 mg/m2/day by continuous IV infusion, Days 1-7; daunorubicin 60 mg/m2/day IV, Days 1, 2, 3, and ixazomib was provided orally at the cohort dose, Days 2, 5, 9, and 12. Consolidation or transplant in this phase was at the discretion of the treating physician. In the second phase (B), induction was administered with the established RP2D of ixazomib in phase A. All patients were to be treated with the following consolidation: cytarabine at 2 g/m2/day, days 1-5 with ixazomib on days 2, 5, 9, and 12 at the cohort dose for consolidation. A classical 3 + 3 patient cohort dose escalation design was used to determine whether the dose of ixazomib could be safely escalated in 3 cohorts (1.5 mg/day, 2.3 mg/day, 3.0 mg/day), initially in induction (phase A) and subsequently in consolidation (phase B). Secondary objectives included rate of complete remission (CR), disease-free survival (DFS), overall survival (OS), and outcome relationship with CD74. Results: Thirty-nine patients were enrolled, including 15 (38%) patients with secondary AML, either with antecedent myeloid malignancy or therapy-related AML. Twenty-six patients (67%) were male, and the median age was 68 years (range 61-80 years). There were no grade 5 toxicities attributed to study drug. Four patients died before day 30 with persistent leukemia, 3 of whom were replaced for assessment of the MTD. Three other patients were not evaluable and were replaced. Nearly all grade 3 and 4 toxicities were hematologic (Table). There was 1 DLT (grade 4 platelet count decrease extending beyond Day 42). There were no grade 3 or 4 neurotoxicity events attributable to ixazomib. Among the 38 patients evaluable for response, 26 achieved CR, and 2 achieved CR with incomplete count recovery (CRi), for a composite remission rate (CCR) of 74%. Among the 15 patients with secondary AML, 9 achieved CR and 2 achieved CRi, for a CCR of 73%. The median OS for the 39 patients is 40.8 months [95% CI, 17.6, Inf]. The 18-month OS estimate was 63% [95% CI, 49-80%]. Of note, 30 patients underwent stem cell transplantation. There was no significant association between level of CD74 expression on blasts at baseline and response. Conclusions: The highest dose level (3 mg) of ixazomib planned for induction and consolidation in this trial has been reached safely and is the recommended phase 2 dose for both. Notably, to date, no grade 3 or 4 neurotoxicity has been encountered. The remission rate for this regimen in this older adult population appears favorable. With ixazomib added in treatment, we saw no association of response rate with level of CD74 in blasts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

STP938, a Selective CTPS1 Inhibitor, Shows Single Agent Activity and Synergy with BCL2 Inhibition in Preclinical Models of Mantle Cell Lymphoma

Background Proliferating tumor cells are dependent on nucleotide synthesis to support DNA replication and RNA synthesis. The homologous CTP synthetases, CTPS1 and CTPS2, play a central role by catalyzing the rate-limiting conversion of UTP to CTP. Interestingly, while most tissues rely on CTPS2, lymphoid cells depend on CTPS1, as observed in rare patients with inherited CTPS1 loss of function manifesting selective B/T cell defects but no non-hematopoietic phenotype (Martin 2014, PMID 24870241). Herein, we investigate inhibition of CTPS1, alone or in combination, as a potential novel therapeutic target in mantle cell lymphoma (MCL). Methods STP938 (Step Pharma) is a highly selective small molecule inhibitor of CTPS1 with >1,300-fold selectivity over CTPS2. In vitro cell proliferation was assessed at 72 hours by CellTiter-Glo. Synergy was quantified by Bliss scores. 3'seq-RNA profiling was performed before and after a 24-hour exposure to STP938; normalized data were subjected to KEGG pathway enrichment analysis. In vivo efficacy was established following subcutaneous transplantation of MCL cells (Z138) into immunodeficient mice (NSG, n=5 per treatment group); treatment was initiated when tumors reached approximately 50 mm3. STP938 was dosed at 30 mg/kg/day subcutaneously days 1-4 of a 7 day cycle; venetoclax at 75 mg/kg/day orally days 2-5 of a 7 day cycle for three cycles. Tumor size was assessed by calipers. Results Analysis of the Achilles CRISPR dataset identified a dependency of human B cell lymphoma cell lines (n=34) on the de novo but not the salvage pyrimidine synthesis pathway. Of note, whilst CRISPR deletion of CTPS1 resulted in a marked proliferation defect, CTPS2 loss had no effect, consistent with the central role for CTPS1 in lymphoid cells established in human genetic studies. Analysis of publicly available data from primary MCL samples (n=122, GSE93291) identified high CTPS1 expression as a predictor of inferior overall survival (P=<0.0001). A selective CTPS1 inhibitor, STP938, was used to evaluate a potential therapeutic role for CTPS1 inhibition. Of 15 MCL cell lines tested, 6 (40%) were highly sensitive to STP938 (IC50 <0.1 μM), 5 (33%) showed intermediate sensitivity (IC50 0.1 - 1 μM) and 4 (27%) were resistant (IC50 >1 μM). Of note, sensitivity to STP938 was independent of known indicators of poor prognosis, such as TP53 mutation, and was independent of resistance to standard of care therapies such as ibrutinib or venetoclax. Pathway analysis of transcriptomic data (n=9 cell lines) identified downregulation of cell cycle pathways in response to STP938 exposure. Directed analysis identified modulation of BCL2-family transcripts, with STP938 inducing increases in BCL2 and BCL2L1 (BCL-XL) expression and a decrease in MCL1 expression (P=0.02, 0.006 and 0.02, respectively; paired t-test). BCL2 increase and MCL1 decrease upon CTPS1 inhibition have been further confirmed at the protein level (24h, 20nM). Given these findings, in vitro combination studies were undertaken with a selective BCL2 inhibitor. STP938 plus venetoclax demonstrated synergy in 5 of 9 MCL cell lines tested (Figure A). Of note, there was no association between synergy and sensitivity to single agent STP938, with two cell lines resistant to single agent STP938 (IC50 3.1 and >10 μM) nonetheless showing synergy with venetoclax. The synergy between STP938 and venetoclax was further investigated in an in vivo model of MCL. Combination therapy was well tolerated. Single agent venetoclax showed no significant inhibition of tumor growth. Single agent STP938 slowed tumor growth to approximately 50% of vehicle control. The combination of STP938 and venetoclax showed strong synergy with more than 80% inhibition of tumor growth for 3 cycles of treatment (Figure B). Conclusions Dependence of tumor cells on elevated rates of nucleotide synthesis has been exploited by agents such as cytarabine. Whilst therapy for hematological cancers is moving away from cytotoxic drugs in favor of targeted agents, inhibition of nucleotide synthesis remains an attractive target. STP938, a highly selective CTPS1 inhibitor, demonstrates single agent activity in vitro and in vivo, and shows strong synergy with the approved BCL2 inhibitor venetoclax. STP938 will enter clinical development for relapsed refractory B and T cell lymphomas in 2022. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multiomics Analysis Confirms Effective Target Engagement for RVU120 - a First-in-Class CDK8/19 Kinase Inhibitor in AML and MR-MDS Patients and Reveals the Mechanism of Action

RVU120 is a first-in-class CDK8/19 kinase inhibitor. The primary objective of an ongoing Phase Ib study of RVU120 in AML and HR-MDS patients is to determine the preliminary safety profile and the recommended phase 2 dose of RVU120 as a single agent. Characterization of pharmacodynamic (PD) effects is a secondary objective of the trial. PD analysis supports the determination of the recommended phase 2 dose, further clinical development and ultimately positioning of this novel class of targeted agents in the treatment strategies for AML and MDS patients. A battery of tests has been used to establish the relationship between pharmacokinetics (PK) and PD activity, confirm the on-target activity of RVU120, and reveal a mechanism of action of CDK8/19 inhibitors in preclinical models. Specific protein binding of RVU120 in leukemic and normal blood cells has been verified by capturing CDK8/19 kinases after a competition with ATP acyl-phosphate biotin-labeled probes. Changes in CDK8- dependent phosphorylation of STAT5 were used as proximal biomarkers and were measured by flow cytometry. Transcriptome changes were used as downstream biomarkers and were determined by the integrated analysis of bulk RNAseq, ChIPseq and ATACseq. The molecular activity of RVU120 was correlated with anti-leukemic activity and surface marker changes. In patients, PD activity of RVU120 was established by measuring changes in pSTAT5 levels, directly in blast cells and ex vivo in established AML cell lines, exposed to plasma collected from patients treated with RVU120. Downstream transcriptomic effects in patients were assessed by RNAseq and phenotypic changes were tracked by flow cytometry surface marker panels. Preclinical profiling indicated effective CDK8/19 target engagement and inhibition of pSTAT5 levels at low nanomolar concentrations of RVU120, in both established cell lines and primary leukemic cells. The antileukemic activity was tightly correlated with levels of biomarker inhibition, however, in a group of super-responders enriched for NPM1 mutants and leukemic stem cells, efficacy was achieved at partial biomarker inhibition levels. Downstream profiling revealed inhibition of homeobox genes and induction of STAT5-dependent genes as two major transcriptomic programs regulated by RVU120. Homeobox genes inhibited by RVU120 are essential for the viability and self-renewal of leukemic cells, whereas many STAT5 -dependent genes are known as master regulators of erythropoiesis. Consistently, treatment of healthy and transformed CD34+ cells with RVU120 results in the commitment to erythropoiesis. Profiling of patient samples largely confirmed preclinical findings. Inhibition of pSTAT5 in CD34+ blasts from AML and MDS patients tightly correlated with achieved exposures after the first dose and at a steady state. At the time of submission pSTAT5 inhibition has already reached >50%, a threshold that based on preclinical predictions is sufficient for robust efficacy in selected groups of super-responder patients. Further increase at higher doses, is expected to result in deeper, more durable responses in broader populations of patients. Broad transcriptomic changes were observed in patients after treatment with RVU120. Notably, potent inhibition of homeobox genes was observed in NPM1 mutant blasts and induction of erythroid master regulator genes was observed in SF3B1 mutant cells with erythroid arrest. Clear hallmarks of erythroid differentiation were further identified in several patients by specific changes in surface markers. These patients achieved meaningful clinical responses such as reduction of blasts and transfusion independence, respectively.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Altering Drp1-Dependent Mitochondrial Fission and Metabolism

Background: Mutational (RAS, PTPN11 etc.) or non-mutational activation of RAS/RAF/ERK pathway is associated with primary or secondary resistance to venetoclax in AML therapy (Zhang et al., Sig Transduct Target Ther. 2022). While upregulation of alternate antiapoptotic proteins e.g. Mcl-1 has been implicated in resistance to venetoclax (Zhang et al., Sig Transduct Target Ther. 2022) role of mitochondrial dynamics (fission/fusion) in venetoclax resistance has not been well explored. Oncogenic RAS activates downstream ERK1/2 phosphorylation and mitochondrial fission through ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1 (Nagdas et al., Cell Rep. 2019). Drp1 deletion inhibits tumorigenesis of KRAS driven pancreatic cancer, but the importance of RAS driven changes in mitochondrial dynamics in AML is poorly defined. ERK1/2 inhibition may therefore provide insights into the dependence of AML cells on altered mitochondrial dynamics and metabolism driven by oncogenic RAS. Objectives: We assessed the synergistic anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and in primary patient samples (n=8). Further, we investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., J Med Chem. 2018) a close analog of ASTX029, which is currently under clinical investigation in solid tumors (NCT03520075), was highly synergistic with venetoclax at inducing apoptosis in AML cells. NRAS-mutant OCI-AML3 and THP1 cells, that are intrinsically resistant to venetoclax, were sensitized to venetoclax by ERK1/2 inhibition with a combination index (CI) of 0.008 (cell death ~90% for combination versus 20% with venetoclax alone). Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax alone (CI=0.6). The combination was also effective in eliminating bulk and CD34+CD38- leukemia initiating cells (LICs) in primary AML samples (CI:0.03-0.23). Colony formation assay and single cell level CyTOF analysis using an NRAS-mutant PDX showed markedly impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to single agent ERK1/2 inhibition and combination treatment, indicating a potential impact on stemness. Mitochondrial reactive oxygen species (ROS) mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML (Schimmer et al., Cell Stem Cell. 2018). As a result of ERK1/2 inhibition, pDrp1 (Ser616) levels decreased, and mitochondrial length (p<0.001) and mitochondrial ROS levels (p<0.01) were increased, suggesting impaired fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Rescue with overexpression of active phospho-mimetic form of Drp1 i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells resulted in decreased apoptosis and reversed the synergy between ERK1/2 and Bcl-2 inhibition when compared to wild type and phospho-null (Ser616Ala) Drp1 overexpression (p<0.001, Fig 1a). Of note, overexpression of Drp1-Ser616Glu-Ser637Ala phospho-mimetic led to decreased mitochondrial length (Fig 1b), suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, decreased maximal oxygen consumption rate (p<0.05) and ATP production (p<0.05), as well as reduced ROS production (p<0.01). Moreover, combination treatment failed to significantly induce ROS production in cells expressing the phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Collectively, these results indicate that inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. Conclusion: ERK1/2 inhibition alters Drp1 dependent mitochondrial dynamics and metabolism, resulting in mitochondrial defects and overcoming resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal