CRISPR tiling screen is a powerful tool to identify protein regions relevant to its biological function. Understanding the functional relevance of the regions of target protein is of great help for structure-based drug discovery. Studying the drug resistance mechanisms of small-molecule inhibitors is important for the development and clinical application of the compounds. Using MEK1 and MEK inhibitors as example here, we demonstrate the utility of CRISPR tiling to identify regions essential for cancer cell viability and regions where mutations are resistant to MEK inhibitors. We study the drug resistance mechanisms of the regions and discussed the potential, as well as limitations, of applying the technology to drug development. Our findings demonstrate the value and prompt the utilization of CRISPR tiling technology in structure-based drug discovery.

There has been renewed interest in pursuing cyclin-dependent kinase 2 (CDK2) as a therapeutic target for cancer treatment, given its essential role in driving the survival of CCNE1-amplified tumors and mediating resistance to CDK4/6 inhibitor treatment in estrogen receptor positive breast cancer. This has resulted in the identification of a next generation of orthosteric inhibitors which have increased selectivity for CDK2 kinase versus other CDK-family members. This study aimed to contrast genetic and chemical perturbation of CDK2 in human tumor cell lines with and without CCNE1-amplification. Methods: Cellular responses were assessed using assays for proliferation (CyQUANT), clonogenicity, cell cycle (flow cytometry), and western blotting. NanoBRET tracer displacement assays were used to examine orthosteric kinase inhibition in cells. Genetic perturbation of CDK2 used siRNA and CRISPR-Cas9 editing with sgRNA. Four CDK2 inhibitors currently undergoing clinical trials were profiled. Results: CDK2-targeting siRNA conferred a >75% decrease in the clonogenicity of CCNE1-amplified OVCAR-3, Kuramochi and FUOV1 cells, thus confirming dependence of the cells on CDK2. The non-CCNE1 amplified cells TYK-nu and PEA2 were not sensitive to CDK2-siRNA. The orthosteric CDK2 inhibitors inhibited cellular CDK2 with IC50 values in the range of 1-20 nM. As expected, the compounds inhibited the proliferation of CCNE1-amplified cell lines but they also demonstrated activity against non-CCNE1 amplified TYK-nu cells (one example had GI50 values of 89 ± 8 nM in OVCAR-3 cells, and GI50 values of 122 ± 13 nM in TYK-nu cells, respectively). Consistent with the role of CDK2 in controlling the G1 checkpoint, we observed G1 arrest in the CCNE1-amplified cell line OVCAR-3 following CDK2 CRISPR knock-out. As expected, CDK2 gene deletion did not significantly impact the cell cycle in the TYK-nu non-CCNE1 amplified cell line. In contrast, an orthosteric CDK2 inhibitor induced a G2 arrest in the TYK-nu cell line at doses 2 - 5-fold greater than the compound GI50. Evidence of cyclin E1 accumulation was observed in both CCNE1-amplified and non-amplified cells following orthosteric CDK2 kinase inhibitor treatment (48 - 72 hours). It was also observed following generation of an F80G CDK2 mutant TYK-nu cell line and treatment with the bulky purine analog 3MB-PP1. In contrast, gene-silencing, or gene-editing of CDK2, had no effect on cyclin E1 levels. Conclusion: We demonstrate that the profiled orthosteric CDK2 inhibitors do not reproduce genetic perturbation of CDK2 in CCNE1-amplified and non-amplified cell lines. We propose the need for more selective CDK2 inhibitors and those that inhibit CDK2 in a manner that more closely reflects the genetic perturbation of CDK2. Joanne M. Munck, Susan J. Tudhope, Kleopatra Papa, Alex Howard, Sam Hogan, Suzanne Kyle, Jessica Watt, Luke Gaughan, Maria Ahn, Stephen R. Wedge, John F. Lyons. Orthosteric CDK2 kinase inhibitors have a distinctive profile when compared to genetic perturbation of CDK2 in CCNE1-amplified and non-amplified tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5331.

Background: Mutational/non-mutational activation of MAPK pathway is a pervasive mechanism of resistance to venetoclax (ABT199) in AML. Our work with ASTX029 and Compound 27, inhibitors of ERK1/2, linked suppressed mitochondrial fission as a mechanism of overcoming MAPK driven resistance to ABT199 in AML. We demonstrated that ERK1/2 inhibition decreased phosphorylation of DRP1, a dynamin like GTPase that drives mitochondrial fission, induced apoptosis in combination with ABT199 in vitro and extended the survival of PDX mouse model of post-ABT199/decitabine relapsed AML (Sharma et al., Blood. 2023). ABT199 resistant AML cells exhibit tighter mitochondrial cristae, resulting in lower cytochrome C release, and enhanced oxidative phosphorylation (OXPHOS). Additionally, gene silencing of BAX and mitochondrial metalloprotease OMA1 were identified as the two top hits that lead to ABT199 resistance (Qi et al., Signal Transduct Target Ther. 2022). Bax activates OMA1 (Jiang et al., PNAS. 2014) and OMA1 cleaves membrane bound long (L-OPA1) to short (S-OPA1) isoform. Steady-state balance of long and short OPA1 isoforms is important for functioning as a molecular staple at cristae junctions. L-Opa1 and S-opa1 oligomerize at cristae junctions to prevent cytochrome C mobilization and release (Varanita et al., Cell Metabolism. 2015). By maintaining tight mitochondrial cristae, Opa1 stabilizes respiratory chain super complexes and inhibits ROS generation. Herein, we investigated the functional aspect of ERK1/2 inhibition on mitochondrial cristae remodeling and metabolism in context of overcoming resistance to ABT199 in AML. Methods: ERK1/2 was inhibited in vitro using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018) and in vivo using ASTX029 (Munck et al., Mol Cancer Ther. 2021), which is currently under clinical trial in solid tumors (NCT03520075). RNAseq, gene set enrichment analysis (GSEA) and CyTOF were performed to identify the mechanism and validated by immunoblotting and flowcytometry. Oxygen consumption rate (OCR) was measured using Seahorse based Mito Stress test. Mitochondrial images were acquired using transmission electron microscopy (TEM) followed by quantification with Imaris. Results: ERKi synergized strongly with ABT199 at inducing apoptosis in RAS mutated and/or ABT199 resistant AML cells and primary samples (p<0.05). Transcriptome profiling revealed that ABT199 upregulated 6 pathways in AML cells: OXPHOS, Myc targets, E2F targets, G2M checkpoint, epithelial mesenchymal transition and KRAS signaling. Interestingly, these pathways were downregulated in response to ERKi+/-ABT199 treatment (FDR<0.05). OPA1, one of the core enrichment proteins for OXPHOS gene set, maintains mitochondrial cristae and contributes to resistance to ABT199 (Chen et al., Cancer Discov. 2019). ERKi in combination with ABT199 induced proteolysis of OPA1 and increased the S-Opa1/L-Opa1 ratio in ABT199 resistant OCIAML2 cells and NRAS mutated OCIAML3 cells. Single cell proteomics using CyTOF showed increased expression of Bax in response to ASTX029+ABT199 in OCIAML3 xenograft. Knockdown of Bax inhibited the proteolysis of Opa1 as well as apoptosis mediated by ERKi+ABT199. Structurally, ERK1/2 inhibition in combination with ABT199 increased cristae width (p<0.0001) with simultaneous release of cytochrome C (p<0.001) as observed by TEM and flowcytometry respectively. Since, ABT199 treatment significantly enriched for OXPHOS, we examined the metabolic effect of ERKi in overcoming ABT199 resistance. Mito Stress test showed increased basal (p<0.0001) and maximal OCR (p<0.0001) and ATP production (p<0.0001) in OCIAML2 ABT199 resistant versus parental cells. Combining ERKi with ABT199 reduced OCR and ATP production in OCIAML3 and OCIAML2 ABT199 resistant cells (p<0.0001). ERKi alone or in combination with ABT199 reduced the activity of electron transport chain complex I (p<0.01). This was accompanied by an increase in mitochondrial membrane depolarization by ERKi+ABT199 (p<0.0001), suggesting mitochondrial dysfunction. Conclusion: Inhibition of ERK1/2, results in mitochondrial dysfunction and mediates Bax dependent proteolysis of Opa1 in AML, which alters mitochondrial cristae width and releases cytochrome C to overcome ABT199 resistance. The findings in this study provide a strong rationale for combining Bcl-2 and ERK1/2 inhibitors as part of an AML treatment protocol.

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination. References: 1. Xue Z, Vis DJ, Bruna A, Sustic T, van Wageningen S, Batra AS, et al. MAP3K1 and MAP2K4 mutations are associated with sensitivity to MEK inhibitors in multiple cancer models. Cell Res. 2018; 28:719-29. Citation Format: Mark Wade, Emiliano Tamanini, Mathieu Unbekandt, Nicola Wallis, John Lyons, Joanne Munck, Andrew Woodhead, Patrick Schopf, Jessie Stow, Charlotte East, Mellissa Clark, Jeffrey St. Denis, Puja Pathuri. Targeting MAPK-driven tumors via inhibition of MAP2K4 [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 5902.

Fold induction in secreted cytokine concentrations in PBMC supernatants after IAP antagonist treatment

Comparison of XIAP activity, cIAP1 activity and selectivity of clinical IAP antagonists

Abstract Clinical efficacy of RAF, MEK1/2 (MEK) and KRASG12C inhibitors has been demonstrated in MAPK-activated cancers. However, response is often short-lived due to resistance mechanisms, which commonly confer reactivation of ERK1/2 (ERK). As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for overcoming resistance to upstream inhibition. ASTX029 is a potent and selective dual-mechanism ERK inhibitor. Due to its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK by MEK. It is currently in Phase 1/2 trial in patients with advanced solid tumors (NCT03520075). Here we demonstrate the potent activity of ASTX029 in models of acquired MAPK inhibitor resistance. Two MAPK inhibitor-resistant models were generated by continuous culture of A375 (BRAFV600E -mutant melanoma) cells in the presence of the BRAF inhibitor vemurafenib (A375R) or by introduction of an NRASQ61K mutation (A375-NRASQ61K). The A375-NRASQ61K and A375R cell lines were resistant to vemurafenib in cell proliferation assays (IC50 >3 µM and >10 µM respectively, compared to IC50 110 nM in parental A375 cells). They were also less sensitive to the MEK inhibitor selumetinib (IC50 270 nM and 1000 nM, respectively, compared to IC50 43 nM in parental A375). Both models were highly sensitive to ASTX029, with proliferation assay IC50 values of 12 nM (A375-NRASQ61K) and 7.2 nM (A375R), values which were not significantly different to the ASTX029 IC50 of parental A375. ASTX029 inhibited MAPK signalling in both models, whereas vemurafenib and selumetinib had no effect. Treatment of A375R tumour-bearing mice with 50 mg/kg bid vemurafenib (which causes significant tumor growth inhibition in parental A375 xenografts), did not result in significant tumor growth inhibition. In contrast, 75 mg/kg qd ASTX029 conferred significant anti-tumor activity (P< 0.001). The acquisition of MEK mutations is a known resistance mechanism to RAF, MEK and KRASG12C inhibitors. We performed a CRISPR-tiling screen on MEK1 and identified mutations that conferred resistance to MEK inhibitors. The identified MEK1 mutations either caused MEK activation (indicated by elevated pERK levels) or were predicted to prevent MEK inhibitor binding (supported by MEK1 structural analysis). All mutations conferred resistance to selumetinib (7- to 200-fold increase in cell proliferation IC50 values relative to parental cells). Mutations predicted to prevent MEK inhibitor binding were sensitive to vemurafenib and the pan-RAF inhibitor LY3009120, whereas those that caused MEK1 activation were resistant (5- to 10-fold increase in cell proliferation IC50 relative to parental cells). However, they were all sensitive to ASTX029 (IC50 values of 4 to 17 nM). We conclude that the MAPK inhibitor resistance mechanisms described did not confer resistance to ASTX029. These data highlight the therapeutic potential of ASTX029 for the treatment of cancers, which have acquired resistance to inhibitors of upstream components of the MAPK pathway. Citation Format: Zhiqiang Zhang, Christopher Hindley, Andrea Biondo, Nicola Wallis, John Lyons, Joanne Munck. ASTX029 is a dual mechanism ERK1/2 inhibitor with activity in models of MAPK-inhibitor resistance [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 B164.

Background: Activation of RAS/RAF/MEK/ERK (MAPK) pathway is common in myeloid malignancies and is a pervasive mechanism of inherent/acquired resistance to targeted inhibition of Bcl-2, mutated IDH and FLT3 in acute myeloid leukemia (AML) (Zhang et al., Sig Transduct Target Ther. 2022). ERK1/2 are terminal kinases in the MAPK pathway and may be suitable targets regardless of upstream mutational/non-mutational mechanisms of MAPK pathway activation. ASTX029 is a dual-mechanism inhibitor of ERK1/2 in clinical development. ASTX029 inhibits catalytic ERK1/2 activity and prevents feedback phosphorylation of MEK1/2, a potential mechanism of resistance. Oncogenic RAS drives ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1, activating mitochondrial fission (Nagdas et al., Cell Rep. 2019). Aims: Therapeutic synergy of venetoclax (Bcl2i) and ASTX029 was evaluated in context of venetoclax resistance. As mitochondria-centered processes contribute to venetoclax resistance, we studied mitochondrial dynamics (fission/fusion) and function (ROS, metabolism) in the context of ERK1/2 inhibition and combined ERK1/2 and Bcl-2 inhibition. Methods: Synergy of Bcl2 and ERK1/2 inhibition was assessed using Annexin V assay followed by RNAseq analysis to identify molecular mechanisms. Results: Compound 27 (ERKi), a close analog of ASTX029 (Munck J et al., Mol Cancer Ther, 2021), was highly synergistic with Bcl2i [combination index (CI) of 0.008] in AML cells with intrinsic (OCI-AML3, RAS mut) and acquired (OCI-AML2 isogenic parental and Bcl2i resistant) resistance to Bcl2i. The observed synergy was also confirmed in primary samples (N=8) (bulk CD45+ and CD34+/38- leukemia initiating cells) obtained from patients with AML previously treated with Bcl2i combinations (CI:0.03-0.23). Mechanistically, the synergy was mediated by altered mitochondrial attributes including increased mitochondrial ROS, decreased Mcl1, and decreased membrane polarization. ERKi treatment resulted in decreased phosphorylation of Drp1(Ser616) and increased mitochondrial length. DRP1 colocalization with mitochondrial outer membrane TOM20 was decreased with ERKi treatment. Mitochondrial imaging with super-resolution microscopy confirmed that overexpression of phosphomimetic Drp1 (Drp1-Ser616Glu-Ser637Ala) resulted in shorter mitochondrial length, unlike phosphonull Drp1, which did not alter mitochondrial length (Fig.1). The apoptotic phenotype associated with synergy of Bcl2i and ERKi was partially reversed with overexpression of either Mcl1 or phosphomimetic Drp1. Transcriptome profiling revealed upregulation of 6 pathways in response to Bcl2i including oxidative-phosphorylation, electron transport chain, Myc targets, E2F targets, epithelial mesenchymal transition, and KRAS signaling. Interestingly, these pathways were downregulated after ERKi+Bcl2i treatment. Other mitochondria relevant transcripts downregulated with ERKi were OPA1, MFN1 (fusion), and MFF (recruits DRP1 to mitochondria). Finally, ERKi treatment alone and in combination with Bcl2i reduced basal oxygen consumption and ATP production in OCI-AML3 and OCI-AML2 Bcl2i resistant cells. In-vivo efficacy studies in NSG mice using RAS mutant AML PDX are ongoing. Summary/Conclusion: ERK1/2 inhibition demonstrates strong synergy with venetoclax and overcomes resistance to venetoclax. This synergy is largely mediated through effects on mitochondrial structural and functional dynamics. Our findings provide a strong rationale for clinical development of ERK 1/2 inhibitor ASTX029 in the treatment of AML and highlights the impact of targeting mitochondrial dynamics.Keywords: Acute myeloid leukemia, Mitochondria, Leukemia

XIAP antagonism in A375 cells in the presence or absence of TNF-α

Supplementary Tables and Figures

Data Supplement from DNA-PK—A Candidate Driver of Hepatocarcinogenesis and Tissue Biomarker That Predicts Response to Treatment and Survival

Preparation of 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one

Supplementary figures 1-7 show the effects of AST029 on ERK signalling in RAS-mutant cell lines (1) and tumour xenograft tissue (3), ASTX029 plasma PK linearity (2), ASTX029 activity in a cell line panel showing MAPK mutation status (4) the effects of ASTX029 on mouse body weight (5), PKPD effects of ASTX029 following b.i.d dosing to Colo205 tumour-bearing mice (6) and PD effects of ASTX029 following qd dosing to A375R tumour-bearing mice (7).

Table listing details of antibodies used

Abstract Background Dysregulation of cell cycle and transcriptional processes promote tumorigenesis and tumor growth. Due to its dual role in regulating both cellular processes, CDK7 is an attractive therapeutic target, with several CDK7 inhibitors in clinical trials. Preclinical data suggests that such agents could have utility across a range of tumor types particularly those driven by defects in the regulation of cell cycle and transcriptional processes. Consequently, potential patient populations are broad and often lack well-defined patient stratification. To identify specific cancers with greater dependence on CDK7 activity, we employed the use of a cell line panel screen coupled with bioinformatic analysis of association of drug sensitivity to molecular features. Materials and Methods To explore tumor sensitivities to CDK7 inhibition we profiled two CDK7 inhibitors (THZ1 and LY3405105; Eli Lilly) and the non-selective kinase inhibitor staurosporine as a control across a panel of 468 human cancer cell lines with varied genetic backgrounds. Activity area was used to define drug responses whilst multi-omics features of cell lines obtained from DepMap were used to define potential molecular features associated with drug response. Differential gene expression between sensitive and resistant cell lines was used to identify expression signatures associated with sensitivity to CDK7 inhibition. Results were validated in vitro by characterisation of cell line models in viability assays, western blots and RT-qPCR. Our findings were further validated in vivo in a NSCLC mouse xenograft model. Results Cell panel data showed a wide range of sensitives to selective CDK7 inhibition (LY3405105) compared to the profile of the less selective THZ1 which had a similar distribution of sensitivity as the control staurosporine. Bioinformatics analysis identified a global c-MYC signature with a significant correlation between CDK7 expression and c-MYC expression across the entire cell panel which confers sensitivity to CDK7 inhibition and was highly correlated in lung cancer. Further analysis of CDK7 inhibitor sensitivity in lung cancer as a function of c-MYC expression found that SCLC and NSCLC had the highest correlation. Using an independent set of cell lines with varying c-MYC levels, we confirmed these findings and the results were further validated in vivo where we observed partial tumor regressions. Conclusions Cell panel data coupled to bioinformatic analysis was used to define potentially sensitive patient populations for CDK7 inhibitors. This work demonstrates in vitro and in vivo activity of selective CDK7 inhibition in SCLC and NSCLC cancer models with high c-MYC levels. Citation Format: Keisha Hearn, Maria Ahn, Luke Bevan, Jessica Brothwood, Charlotte East, Anna Esteve-Arenys, Lynsey Fazal, Christopher Hindley, Sabrina James, Justyna Kucia-Tran, John Lyons, Joanne Munck, Harpreet Saini, Mathieu Unbekandt, Dhaval Varshney, Steve Wagner, Andrew Woodhead, Nicola Wallis. Identifying sensitive patient populations for CDK7 inhibitors using cell panel screens and bioinformatic approaches [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 A110.

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.

Supplementary tables 1-6 summarise the kinase panel screen data (1) mouse PK parameters (2) and further details of the anti-tumour activity (5) conferred by ASTX029, plus details of cell lines used in this study (3 and 4) and further details of the effects of ASTX029 and other MAPK inhibitors in models of MAPK resistance (6).