Med Chem Research Articles

P1063 Impact of Upadacitinib in Crohn’s Disease in the Medium Term: Results from the UPITA-Crohn Registry in Clinical Practice

Abstract Background There is still insufficient evidence regarding the effectiveness and safety of Upadacitinib in real-world settings. This recently approved oral JAK inhibitor for moderate-to-severe Crohn’s disease is the focus of this study, which aims to evaluate them in this clinical context. Methods Clinical and biochemical data were retrospectively collected from 107 patients who initiated treatment with Upadacitinib across 10 hospitals in Andalusia. Clinical remission (CR) was defined as a Harvey-Bradshaw score of less than 5, clinical and biochemical remission (CBR) as a Harvey-Bradshaw score <5, PCR <5 mg/L, and Calprotectin <250 μg/g, and remission without steroids (RWS) as a Harvey-Bradshaw score <5 with no corticosteroid use since week 12. These and other variables were evaluated at week 12 and at 6 months. An intention-to-treat analysis was performed. Results The mean age of the 107 recruited patients was 41 years (range: 18–76), with 59% being female. Disease involvement was ileocolic in 52%, ileal in 33%, and colonic in 15%. Among these patients, 25% were smokers and 15% were ex-smokers. Extraintestinal manifestations were present in 45%. The mean disease duration was 13 years (range: 1–40), and 35% had previously undergone resective surgery. The mean number of failed advanced treatments was 3. At treatment initiation, 11% were on 5-ASA therapy, 10% on immunomodulators, and 33.6% on corticosteroids. Induction with 45 mg was administered for 8 weeks in 12.2% of patients, for 12 weeks in 75.5%, and for 16 weeks in 12.2% (Table 1a). Clinical and biochemical efficacy data are shown in Table 1b. Among the patients who completed 12 weeks of treatment (84 patients), 54.6% achieved CR, and 18.5% achieved CBR. At 6 months (43 patients), 47.4% achieved CR, 22% achieved CBR, and 42.3% achieved SFR. At the time of the study, 16 patients (14.9%) had discontinued treatment before the 6-month mark, primarily due to primary nonresponse (68%). One patient died from complications of the disease. Other reasons for discontinuation included popliteal thrombosis, pregnancy, fever, and a respiratory infection, each affecting one patient. Several mild and transient adverse effects were reported (5% of all patients), including 2 cases of acne, 2 of elevated liver transaminases, and 2 infections. Conclusion Upadacitinib is a favorable, effective, and safe treatment in the short to medium term for a cohort of patients with refractory Crohn’s disease. References -Clark JD, Flanagan ME, Telliez JB. Discovery and development of Janus kinase (JAK) inhibitors for inflammatory diseases. J Med Chem 2014; 57: 5023–38. -Padda IS, Bhatt R, Parmar M. Upadacitinib. 2023 Jun 3. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan–. PMID: 34283454. -Loftus EV Jr, Panés J, Lacerda AP, Peyrin-Biroulet L, D’Haens G, Panaccione R, Reinisch W, Louis E, Chen M, Nakase H, Begun J, Boland BS, Phillips C, Mohamed MF, Liu J, Geng Z, Feng T, Dubcenco E, Colombel JF. Upadacitinib Induction and Maintenance Therapy for Crohn’s Disease. N Engl J Med. 2023 May 25;388(21):1966-1980. doi: 10.1056/NEJMoa2212728.

JAK3 Inhibitors: Covalent and Noncovalent Interactions of a Cyanamide Group Investigated by Multiscale Free-Energy Simulations.

Janus kinase type 3 (JAK3), an emerging target for treating autoimmune diseases, possesses a front pocket cysteine that is targeted by covalent modifiers, best represented by the marketed drug ritlecitinib (1). Recently, 2,3-dihydro-1H-inden-1-ylcyanamides have been developed as novel JAK3 inhibitors. Among them, the N-(6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide inhibitor (2) and its methylated analogue (3), while being potent inhibitors, displayed different mechanisms of action (covalent vs noncovalent) and binding modes (Casimiro-Garcia et al., J Med Chem 2018). Prompted by this intriguing behavior, we applied a multiscale approach to characterize the reaction mechanism between the JAK3 front-pocket Cys909 and cyanamide-based inhibitors. Quantum mechanics/molecular mechanics simulations showed that 2 can readily form an isothiourea adduct with the Cys909 only when a conserved water molecule assists the reaction as a proton shuttle and that methylation of the 2,3-dihydro-1H-inden-1-ylcyanamide moiety of 2 hampers the isothiourea formation by displacing this water molecule. Metadynamics and thermodynamic integration simulations were applied to investigate the relative abundance of alternative poses accessible to 2,3-dihydro-1H-inden-1-ylcyanamides, explaining the effect of methylation on the relative binding mode preference. This multiscale approach provides new chemical insights into the mechanism of action of cyanamide inhibitors and emerges as an effective protocol to investigate the interaction between drugs and molecular targets.

A new antibacterial with anti-inflammatory properties promotes wound healing through inhibiting cGAS/STING/NF-κB/IRF3 pathway

Benzothiazole-urea hybrid 8l was found to be a potent anti-bacterial agent against methicillin-resistant Staphylococcus aureus (MRSA2858) (MIC = 0.78 μM, Eur J Med Chem. 2022,236:114333). Herein, 8l was further evaluated to remedy the MRSA-infected scald with bacterial infection and severe inflammation. In scalded skin model with MRSA infection, 8l not only effectively reduced bacterial load, but also decreased pro-inflammatory cytokines secretion and promoted collagen deposition to effectively reverse the progression of wound infection and inflammation by blocking cGAS/STING/NF-κB/IRF3 signaling pathway. In vitro model of RAW264.7 cells verified that 8l can inhibit MRSA-induced inflammation via regulating this pathway. All in all, dual anti-bacterial and anti-inflammatory agent 8l could heal MRSA-infected refractory scald by regulating cGAS/STING/NF-κB/IRF3 pathway.

A small molecule compound 759 inhibits the wnt/beta-catenin signaling pathway via increasing the Axin protein stability.

Wnt/β-catenin signaling plays important role in cancers. Compound 759 is one of the compounds previously screened to identify inhibitors of the Wnt/β-catenin pathway in A549 cells [Lee et al. in Bioorg Med Chem Lett 20:5900-5904, 2010]. However, the mechanism by which Compound 759 induces the inhibition of the Wnt/β-catenin pathway remains unknown. In our study, we employed various assays to comprehensively evaluate the effects of Compound 759 on lung cancer cells. Our results demonstrated that Compound 759 significantly suppressed cell proliferation and Wnt3a-induced Topflash activity and arrested the cell cycle at the G1 stage. Changes in Wnt/β-catenin signaling-related protein expression, gene activity, and protein stability including Axin, and p21, were achieved through western blot and qRT-PCR analysis. Compound 759 treatment upregulated the mRNA level of p21 and increased Axin protein levels without altering the mRNA expression in A549 cells. Co-treatment of Wnt3a and varying doses of Compound 759 dose-dependently increased the amounts of Axin1 in the cytosol and inhibited β-catenin translocation into the nucleus. Moreover, Compound 759 reduced tumor size and weight in the A549 cell-induced tumor growth in the in vivo tumor xenograft mouse model. Our findings indicate that Compound 759 exhibits potential anti-cancer activity by inhibiting the Wnt/β-catenin signaling pathway through the increase of Axin1 protein stability.

Abstract 3350: The pan-quadruplex drug QN-302 targets up-regulated genes in pancreatic cancer and in other cancer types and correlate with poor patient prognosis

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for clinical evaluation and is currently in a Phase 1 clinical trial at select oncology centers in the USA. Genes that are significantly down-regulated by QN-302 are mostly up-regulated genes in human PDAC. We report here that some of these genes are also up regulated in other cancer types, whose expression frequently correlates with poor patient prognosis. We have used human cancer data from https://www.proteinatlas.org which contains pathology information based on mRNA and protein expression data from 17 different forms of human cancer, together with millions of in-house generated immunohistochemically stained tissue sections, images and Kaplan-Meier plots showing the correlation between mRNA expression of each human protein gene and cancer patient survival. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells exposed to QN-302 for 24 hrs. (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their gene products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Members of the PDAC susceptible gene panel are mostly disfavored in prostate, lung and breast cancer. However, the patient data does not discriminate between cancer sub-types nor does it show resistant data. They are especially favored in colorectal, liver and renal cancers, suggesting that QN-302 may have anticancer effects in these cancers as well as in PDAC. We also do not exclude the strong possibility that each cancer type also has its own group of G-quadruplex containing genes that are sensitive to QN-302. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 targets up-regulated genes in pancreatic cancer and in other cancer types and correlate with poor patient prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3350.

Abstract 576: The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for Phase 1a clinical trials in USA and is currently under clinical evaluation. RNA-seq whole-genome transcriptome analysis revealed that QN-302 selectively targets G-quadruplexes in cancer-related genes, notably by forming stable G-quadruplex complexes with appropriate promoter sequences. This results in the transcriptional down regulation of affected genes, which map to for example, the hedgehog, axon guidance, WNT/β-catenin and P38 signal transduction pathways. In many instances individual expression changes are modest, though statistically significant: cumulatively, they affect whole pathways. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their genes products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Cellular transcriptome data on these eleven genes is presented here, which together with earlier reports on them, supports the designation of QN-302 as a pan-G-quadruplex agent targeting a group of major genes involved in PDAC maintenance and metastasis. Data from a xenograft model for pancreatic cancer is also available for four of these genes, which concurs with the cellular data. We suggest that the identification of these 11 genes may also provide a basis for biomarker selection and possible patient stratification based on transcriptome data. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 576.

Abstract 4587: Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models

Abstract Background: The Proto-oncogenic PIMs family are nuclear and cytoplasmic Ser/Thr kinases (PIM1, PIM2, and PIM3) that are constitutively active and play a vital role in proliferation and survival in Multiple Myeloma (MM). Activated PIM1 kinase can induce progression of the cell cycle, inhibition of apoptosis, and modulation of other signal transduction pathways. Methods: BLX-0631 and a few analogs evaluated against a panel of MM cell lines (EJM, IM-9, L-363, LP-1, MM-1R, MM.1S, MOLP-2, NCI-H929, OPM-2, RPMI8226 and U-266) and primary MM patient samples by CellTiter-Glo® (CTG) assays. Cellular apoptosis, Cell cycle analysis, Flow Cytometry, and bone marrow microenvironment experiments on the most sensitive cells were performed. PIM1 and downstream targets by western blot, cell migration, and invasion on our lead PIM1 inhibitor BLX-0631 along with, kinome selectivity, ADME-Tox, PK, and in vivo MM.1S efficacy experiments were conducted. Results: The application of our empirical MolecuLernTM fragment library, PIM1 crystal structure, scaffold hopping, and the enumeration features of MolecuLernTM in combination with 3, 6, and 7-group analysis on pyridazine, lead to a synthesizable potential new lead series, representing Med Chem tractability and developable characteristics. Further, the consensus scoring, binding energies, and MolecuLern tractability filters were applied and led to the discovery of the BLX-0631 series that inhibits the PIM1 kinase. The active compounds, with their binding modes and lead optimization strategies, lead to the synthesis and testing of over 60 novel entities as PIM1 kinase inhibitors. Thus, based on our previous chemotype series and scaffold hopping method, the impact of changing the 3, 6, and 7 positions, while retaining the pyridazine core led to the discovery of BLX-0631 and its series of compounds exhibiting <5 nM potency. BLX-0631 in CTG assay demonstrated 100% cell growth inhibition across all MM cell lines tested. The lead compound BLX-0631 is a very potent inhibitor with an IC50 of 5.0/17/5.0 nM when tested against PIM1, PIM2, and PIM3 kinases respectively, and selective against a panel of 468 kinase panels. Dosing through IV and PO delivery routes, formulations, salt forms of BLX-0631 in mice, PK properties along with ADME-Tox, P450, and hERG results will be presented. In vivo MM.1S mouse xenograft models of BLX-0631 at 15, 30 and 45 mg/Kg, p.o, QD demonstrated TGI of 16.28%, 31.81% & 35.16% dose-dependently. Conclusions: The present study demonstrates that PIM1 plays a vital role in the proliferation and survival of Multiple Myeloma. BLX-0631 induces full inhibition of all MM cell lines, suppresses cancer cell proliferation in vitro, and exhibits promising efficacy in MM tumor models. BLX-0631, a nominated IND candidate can serve as a novel targeted agent for treating Multiple Myeloma patients. Citation Format: Zhaoang li, Chenyu Lin, David J. Bearss, Hariprasad Vankayalapati. Development of highly selective, potent, orally available PIM1 inhibitor BLX0631 shows a therapeutics potential in multiple myeloma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4587.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1634.

Abstract 1508: Membrane heparan sulfate proteoglycans work with cadherins to establish the directional orientation of migrating cancer cells

Abstract A role of heparan sulfate proteoglycans (HSPGs) in cancer cell differentiation, proliferation and migration has been recognized and related to their ability to specifically interact with growth factors, morphogens, and extracellular matrix proteins, mainly through sulfated groups on their glycosaminoglycan (GAG) chains. Due to the considerable diversity of HSPGs and of their GAG chains, and to the lack of specific ligands, a precise molecular characterization of the biological role of HSPGs in cancer cells is still lacking. HSPGs, besides working as coreceptors for growth factors and morphogens, may have a determinant and autonomous role in cancer signaling events, regulating cell adhesion, migration, and invasiveness. Being overexpressed and over sulfated in cancer cells, HSPGs may become attractive tumor targets for cancer diagnosis and therapy. We had used the tetra-branched peptide NT4, which binds sulphated GAG chains of HSPGs, to validate HSPGs as potential tumor associated antigens in different human solid tumors. The same NT4 peptide had been tested as a tumor targeting agent for either cancer cell imaging or therapy, in vitro and in vivo [1]. Once identified the sulfated GAG specificity of NT4, the peptide has also been used as a specific tool for studying the role of HSPGs in cancer cell migration and invasiveness, with the aim of proposing HSPG as potential drug targets for interfering with cancer invasiveness and metastatic potential [2,3]. We found that NT4 inhibits adhesion, oriented migration, and colony formation in different human cancer cell lines. We investigated the role of HSPGs in migration of different human cancer cell lines, displaying either single-cell mesenchymal migration (PANC-1 pancreas adenocarcinoma, and MDA-MB-231 breast adenocarcinoma) or collective migration (MCF-7 breast adenocarcinoma). After assessing the expression of HSPG and of E- and N- cadherin in each cell line, their specific cellular distribution was detected by confocal microscopy in migrating cells, in wound healing experiments. We found that distribution of either E- or N- cadherins on the membrane of migrating cells is complementary to that of HSPG. This is particularly clear in collective migration, where HSPGs are exclusively located on the migration front of leader cells, whereas cadherins are only expressed by follower cells. Our results suggest that HSPG may have a crucial role in defining the front of migrating cells, while cadherin-mediated cell-cell contacts on the opposite site, may contribute to inducing protrusion formation at the front of migrating cells, as already suggested [4]. [1]- Brunetti J et al. Sci. Rep. 2015, 5, 17736; [2]- Brunetti J et al. Sci. Rep. 2016, 6, 27174; [3]- Depau L et al. J Med Chem 2020, 63, 15997; [4]- Grimaldi C et al Nat Commun 2020, 11, 5397 Citation Format: Lorenzo Depau, Jlenia Brunetti, Chiara Falciani, Marta Garfì, Maria Francesca Paolocci, Alessandro Pini, Luisa Bracci. Membrane heparan sulfate proteoglycans work with cadherins to establish the directional orientation of migrating cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1508.

P143 Efficacy of the oral tyrosine kinase 2 (TYK2) inhibitor TAK-279 in two preclinical mouse models of colitis

Abstract Background Tyrosine kinase 2 (TYK2), a member of the Janus kinase (JAK) family, is required for the downstream signalling of interleukin (IL) 12 and 23, and interferons (IFN) α/β, which are involved in the pathogenesis of several autoimmune diseases, including inflammatory bowel disease (IBD).1 JAK inhibitors are approved for treatment of IBD, but their dosing levels may be limited due to adverse effects related to JAK1–3 inhibition. TAK-279 is a highly-selective, oral, allosteric TYK2 inhibitor, which binds to the JAK homology 2 pseudokinase domain of TYK2, but not JAK1–3.1,2 This study assessed the efficacy of TAK-279 in two IL-23-dependent preclinical mouse models of colitis induced by (i) adoptive T-cell transfer (TCT) and (ii) anti-CD40 monoclonal antibody (α-CD40 mAb). Methods In the TCT colitis model, CD4+CD45RBhi T cells were transferred to severe combined immunodeficient mice at Day 0, and disease progression followed for 48 days. In the α-CD40 colitis model, T- and B-cell-deficient Rag2-/- mice were challenged with agonistic α-CD40 mAb at Day 0, and disease severity analyzed on Day 7. Mice were divided into four groups per model (n = 12–16 and n = 10 per group in the TCT and α-CD40 models, respectively) to receive vehicle (negative control) or TAK-279, orally, twice-daily, at doses intended to provide either 24hr IC90 (high dose) or IC50 (low dose) coverage, or once-weekly intraperitoneal α-IL-12/23 p40 mAb (positive control). At the end of the treatment period, colonic tissue was obtained; colon weight:length (CWL) ratio, total histology score (THS) and transcriptomic analysis were used to assess efficacy and mechanism of action. Statistical significance of differences between groups was determined using analysis of variance with Tukey’s post hoc test. Results In both models, TAK-279 was maximally efficacious at the IC90 dose; CWL ratio and THS were significantly reduced relative to vehicle and the response was comparable with α-IL-12/23 p40. IC50 dosing was not sufficient in the α-CD40 model and IC90 dosing was required for significant efficacy (Figure). Transcriptomic analysis showed TAK-279-dependent downregulation of genes associated with cytokine signalling, including those related to T-helper 17 cell phenotypes and the IFN α/β pathway. Conclusion TAK-279 was efficacious in two preclinical mouse models of colitis and induced relevant cytokine signalling changes; IC90 coverage provided maximal efficacy in both models. These results support the potential role of potent and selective TYK2 inhibition for the treatment of IBD, where TAK-279 is expected to achieve IC90 coverage in humans. 1. Leit S et al. J Med Chem 2023;66:10473–96. 2. Gangolli EA et al. STAT 2022;1:5.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract A026: Synthesis and structure-activity studies of DNA- and RNA-binding nucleopeptides with a cell penetrating potential

Abstract In the field of modern biomedicine, significant progress has been made in countering or restoring faulty genes using exogenous nucleic acids or their mimetics (NAMs). This approach enables targeted and long-lasting therapeutic effects by circumventing the need of interventions at a protein level. Despite the boom of clinical approvals, the development of NAMs still faces crucial challenges, including susceptibility to metabolic breakdown, limited uptake by cells, self-aggregation, and the potential for immune reactions. In addition to employing advanced delivery strategies, alternative methods to address these issues involve modifying NAMs chemically or combining oligonucleotides (ONs) with cellular carriers, including cell-penetrating peptides. Against this background, herein we present the nucleopeptides (NPs) as a family of unexplored NAMs that bridge the gap between ONs and peptide structures. By incorporating two consecutive amino acids—one bearing a nucleobase on the side chain and the other remaining unaltered—we created a monomer that spans the length of a single nucleotide. This functional module possesses nucleobase recognition elements along with additional functional groups eligible to modulate physio-chemical properties. We investigated this class of macromolecules by preliminarily devising an efficient solid-phase peptide synthesis technique profitable for homo- and hetero-sequences and sparing the monomer assembly in solution. [1] The influence of the amino acid composition on target binding properties, metabolic stability, and cellular penetration, were assessed through the synthesis of a series of homothymine hexameric derivatives. This simplified model was also used to investigate the distance between the nucleobase and the backbone, the absolute configuration of the nucleobase- bearing element, and the polycationic/anionic nature of the unmodified amino acid. [2-4] We evaluated the recognition and the thermodynamic stability of NPs/DNA and NPs/RNA hetero-duplexes through circular dichroism and UV spectroscopy experiments, while we employed confocal microscopy with suitable FITC- labeled derivatives to determine the intracellular localization in target cells. [1,3] Our results suggested that NPs exhibit distinct binding properties towards complementary RNA and DNA sequences according to their aminoacidic composition, and are characterized by a higher affinity compared to ONs and PNAs, thus arising as promising cell penetrating NAMs. [1] Mercurio ME, Tomassi S, et al. J Org Chem. 2016, 81(23), 11612- 11625. [2] Tomassi S, Ieranò C, et al. Bioorg Med Chem. 2018, 26(9), 2539-2550. [3] Tomassi S, Montalban FF, et al. Symmetry 2019, 11(4), 567-582. [4] Tomassi S, Ieranò C, et al. Int J Mol Sci. 2022, 23(15), 8504-8518. Citation Format: Stefano Tomassi, Caterina Ieranò, Stefania Scala, Anna Messere, Salvatore Di Maro. Synthesis and structure-activity studies of DNA- and RNA-binding nucleopeptides with a cell penetrating potential [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A026.

Trial in Progress: A Phase 1b Study of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Patients with Previously Treated Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma or Non-Hodgkin Lymphoma

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited by toxicity related to off-target kinase inhibition and acquired resistance due to BTK C481 mutation. AS-1763 is a potent, highly selective, orally available, and non-covalent BTK inhibitor, equipotent against both wild-type and C481S-mutated BTK with sub-nanomolar IC50 values (Kawahata et al. J Med Chem 2021; 64,14129-14141). In a Phase 1 single-ascending dose study in healthy volunteers, AS-1763 was well tolerated and safe at doses of 5-600 mg and achieved maximum inhibition of B cell activation at doses of 100 mg and above at 1-2 h post-dose and the duration of inhibitory effect increased with dose [Arimura et al. AACR 2022; Cancer Res 2022;82(12_Suppl)]. In preclinical studies, AS-1763 demonstrated a broad spectrum of inhibition against BTK mutations that confer resistance to other non-covalent BTK inhibitors in addition to the C481 mutation. Study Design and Methods: This study is an open-label, multi-center, Phase 1b study of oral AS-1763 in patients with advanced chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) and other B-cell non-Hodgkin lymphomas (NHLs) who have failed or are intolerant to at least two prior lines of systemic therapy. Prior therapy with a covalent BTK inhibitor is permitted; prior use of a noncovalent BTK inhibitor is excluded. This study consists of two parts, i.e., dose escalation and expansion parts. The dose escalation part will follow a 3+3 design with a starting dose of 100 mg BID. Each cycle will be 28 days. Three dose levels will be selected for dose expansion part based on a comprehensive review of data from dose escalation. The dose expansion part consists of 2 cohorts, i.e., Cohort 1 for CLL/SLL and Cohort 2 for B-cell NHL. In each cohort, the first 30 patients will be allocated to the 3 dose levels (10 patients for each), then the same provisional recommended Phase 2 Dose (RP2D) will be selected for Cohort 1 and Cohort 2 after 30 patients have enrolled in either cohort. After the provisional RP2D is selected, up to 28 patients in Cohort 1 and up to 15 patients in Cohort 2 may be treated at the provisional RP2D. In this study, up to 110 patients will be enrolled. Treatment will continue for 24 cycles or until disease progression, occurrence of unacceptable toxicity or discontinuation because of other reasons. Eligible patients include CLL/SLL, Waldenstrӧm macroglobulinemia (WM), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and follicular lymphoma (FL). Patients with transformed disease (e.g., Richter's transformation) prior to or during screening are not eligible. Key exclusion criteria include CNS involvement by systemic lymphoma, stem cell transplant or CAR-T therapy <30 days, and clinically significant cardiovascular disease. The primary objective of the dose escalation part is to determine the maximum tolerated dose/dose-limiting toxicity. Key secondary endpoints include the evaluation of safety profile and tolerability, pharmacokinetics properties, and preliminary anti-tumor activity based on overall response rate (ORR) by investigator according to iwCLL 2018 for CLL, IWW6 for WM, and Lugano Treatment Response Criteria for MCL, MZL, SLL and FL. The primary objective of the dose expansion part is to assess the preliminary anti-tumor activity based on ORR by the Safety Monitoring Committee (SMC). Key secondary objectives are to investigate safety, tolerability, and pharmacokinetic profiles and to assess the preliminary antitumor activity based on ORR by investigator and best overall response, duration of response, progression-free survival and overall survival by investigator and the SMC. This trial will evaluate several key exploratory objectives including evaluation of minimal residual disease negativity, characterization of patient subsets defined by mutation status of BTK and phospholipase C gamma 2 (PLCG2), and characterize BTK and PLGC2 gene mutation before and after disease progression. Changes in the level of biomarkers such as CCL3 and CCL4 chemokines and B-cell receptor pathway signaling will be evaluated prior to, during therapy, and at time of disease progression. The RP2D will be determined based on all the data generated in the study. The study has been registered on ClinicalTrials.gov (NCT05602363).

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Direct, Potent, and TP53-Independent Activity of Sting Agonists Against Acute Myeloid Leukaemia Enhances Venetoclax Efficacy In Vivo

Introduction TP53 mutation occurs in approximately 20% of patients with newly diagnosed acute myeloid leukaemia (AML). Treatment outcomes are universally poor and discovery of new therapies with TP53 independent activity represents an area of very high unmet need. Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) sense cytoplasmic double-stranded DNA to elicit type I interferon production. Direct pharmacologic activation of STING has been investigated in clinical trials with the goal of enhancing immunogenicity. During apoptosis, activated caspases have been shown to suppress activation of the cGAS/STING pathway to render cell death immunologically silent. Whilst the canonical role of STING is stimulation of an inflammatory response, we now show that direct activation of STING by potent and clinically relevant agonists can induce apoptosis directly in cancerous cells by leveraging the high levels of expressed STING in a variety of blood cancers. Importantly, apoptosis induced by STING agonists may be enhanced through combination with diverse BH3-mimetic drugs in a TP53 independent manner (Diepstraten et al, submitted). In this abstract, we highlight the activity of STING agonists in AML and its potential for clinical testing, especially among patients with TP53 mutated disease. Methods We tested the clinically relevant small molecule STING agonist diABZI (purchased from SYNthesis Med Chem), which is currently in early phase clinical trials (NCT03843359, NCT05424380). diABZI was tested alone or in combination with the BCL-2 specific BH3 mimetic drug venetoclax (purchased from Chemgood). Drug sensitivity assays were performed using human AML derived cell lines and primary bone marrow or peripheral blood mononuclear cells from patients with AML or myelodysplastic syndrome with excess blasts 2 (MDS-EB2). Cells were cultured for 48 hours, and cell death assessed by PI or DAPI staining. In vivo experiments were conducted by transplanting NOD- scidIL2Rgamma null (NSG) mice with MOLM-13 TP53 wildtype (WT) or TP53 knockout(KO) cells. Mice then received either no treatment (control), diABZI alone (1.5 mg/kg intravenously twice a week), venetoclax alone (50 mg/kg orally Monday-Friday), or combination diABZI and venetoclax for two weeks ( n=5 per treatment arm). Survival analysis was performed using the log-rank (Mantel-Cox) test with Bonferroni correction. Results Single agent diABZI was active in 5/6 human AML cell lines tested, including cell lines with TP53 mutation. Isogenic MOLM-13 TP53 KO cells demonstrated the same sensitivity as the TP53 WT parental cells. BAX/BAK KO and STING KO cell lines were resistant to diABZI, confirming that cell death occurred by apoptosis being dependent on BAX/BAK as well as STING. Combination of diABZI and venetoclax resulted in additive killing in MOLM-13 and OCI-AML3 cells (Bliss scores of 7.62 and 8.06, respectively). Ex vivo testing was performed on 18 different primary specimens (16 AML and 2 MDS-EB2; 12/18 cases had high risk genetic or clinical features such TP53 mutation, complex karyotype, secondary disease, or relapse after prior therapy) (Figure 1). Strikingly, diABZI was active as a single agent in 16/18 cases, with marked efficacy in 11 cases (IC 50 <100 nM). The addition of venetoclax enhanced killing of AML cells, especially in cases resistant to STING agonist alone. In vivo, diABZI monotherapy and combination therapy with venetoclax was well tolerated without significant weight loss or hematologic toxicity. Treatment with diABZI prolonged survival in the highly aggressive MOLM-13 xenograft model (Figure 2). In both the TP53 WT and TP53 KO context, single agent diABZI was superior to single agent venetoclax ( p=0.012 and p=0.011, respectively), but survival was most prolonged in mice receiving combination therapy ( p=0.013and p=0.012, respectively). Conclusion Activation of STING can exert direct anti-tumour effects in blood cancers via induction of apoptosis. Human AML cells are highly sensitive to the pro-apoptotic effects of STING agonists. The combination of venetoclax and STING agonist shows marked efficacy in ex vivo and in vivo studies, including in models of TP53 defective AML. Combining STING agonists with venetoclax represents a highly promising and novel therapeutic approach for AML and should be considered for accelerated early phase clinical trials.

Reply letter to Dr. Wojtasek regarding: Oxidation of flavonoids by tyrosinase and by o-quinones–comment on "Flavonoids as tyrosinase inhibitors in in silico and in vitro models: basic framework of SAR using a statistical modelling approach” published by K. Jakimiuk, S. Sari, R. Milewski, C.T. Supuran, D. Söhretoglu, and M. Tomczyk (J Enzyme Inhib Med Chem 2022;37:427-436)

Reply letter to Dr. Wojtasek regarding: Oxidation of flavonoids by tyrosinase and by o-quinones–comment on "Flavonoids as tyrosinase inhibitors in in silico and in vitro models: basic framework of SAR using a statistical modelling approach” published by K. Jakimiuk, S. Sari, R. Milewski, C.T. Supuran, D. Söhretoglu, and M. Tomczyk (J Enzyme Inhib Med Chem 2022;37:427-436)

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ADVERTISEMENT RETURN TO ISSUEPREVArticleIssue Publication InformationCite this: ACS Bio Med Chem Au 2023, 3, 2, XXX-XXXPublication Date (Web):April 19, 2023Publication History Published online19 April 2023Published inissue 19 April 2023https://doi.org/10.1021/bgv003i002_1676063Copyright © 2023 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views-Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (221 KB) Get e-Alerts Get e-Alerts

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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIssue Editorial MastheadCite this: ACS Bio Med Chem Au 2023, 3, 2, XXX-XXXPublication Date (Web):April 19, 2023Publication History Published online19 April 2023Published inissue 19 April 2023https://doi.org/10.1021/bgv003i002_1676064Copyright © 2023 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views-Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (200 KB) Get e-Alerts Get e-Alerts

Abstract 3860: ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism

Abstract Background: Primary or secondary resistance to venetoclax is pervasively associated with mutational/non-mutational activation of RAS/RAF/ERK pathway which confers metabolic alterations as a compensatory adaptation. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition followed by RNAseq analysis to identify molecular mechanisms responsible for overcoming resistance to venetoclax. Results: ERK1/2 inhibition using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029, was highly synergistic with venetoclax at inducing apoptosis in AML. NRAS-mutant OCI-AML3 and THP1 cells, that are inherently resistant to venetoclax, were sensitized to venetoclax when combined with ERKi with ~90% apoptosis compared to 20% by venetoclax alone (Combination Index = 0.008). Synergy was also confirmed using isogenic OCI-AML2 cells with lab-generated acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax (CI=0.6). Leukemia progenitor cells in primary AML samples were also depleted by the ven+ERKi combination (CI:0.03-0.23). In line with our in-vitro findings, treatment with ASTX029 as a single agent or in combination with venetoclax significantly inhibited leukemia burden and increased survival in an OCIAML3 xenograft model (p<0.05). Single agent ERKi and combination treatment using an NRAS-mutant PDX resulted in significantly impaired clonogenic potential and decreased expression of cMyc and CD44 in CD34+CD38- population as demonstrated by CyTOF, indicating depletion of progenitors. Transcriptomic profiling of OCIAML3 cells revealed upregulation of 6 pathways in response to venetoclax including oxidative phosphorylation (OXPHOS), electron transport chain, Myc targets, E2F targets, epithelial mesenchymal transition and KRAS signaling. Interestingly, these pathways were downregulated after ERKi+venetoclax treatment along with decrease in glycolysis and fatty acid metabolism (FDR<0.05). Since, venetoclax treatment significantly enriched for OXPHOS, we examined the metabolic effect of ERKi to overcome venetoclax resistance. Seahorse analysis showed increased basal and maximal respiration, and ATP production in OCIAML2 venetoclax resistant versus parental cells. Combining ERKi with venetoclax reduced OCR and ATP production in OCIAML3 and OCIAML2-venetoclax resistant cells. This was accompanied by an increase in membrane depolarization (p=0.001) as well as enhanced mitochondrial ROS (p<0.001). Conclusion: The inhibition of ERK1/2 alters mitochondrial metabolism and functional integrity to overcome 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. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Christopher Hindley, Kim-Hien Dao, Martin Sims, V. Lokesh Battula, Michael Andreeff, Gautam Borthakur. ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism. [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 3860.