Cellular Target Engagement Research Articles

Abstract 5551: Potent oral ERAP1 inhibitors modify the immunopeptidome in vivo and are novel immunotherapy agents

Abstract Immune checkpoint therapy has changed the cancer treatment paradigm, however the majority of patients respond poorly to T cell checkpoint blockade alone. Emerging evidence suggests factors that increase the diversity of the immunopeptidome such as high tumor mutation burden and HLA-I evolutionary divergence (HED), improve clinical response to checkpoint blockade providing an impetus to develop strategies that increase the presentation of cancer associated antigens to the immune system. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an aminopeptidase that trims peptides loaded into classical and nonclassical MHC molecules. Human genome-wide association studies have identified single nucleotide polymorphisms within ERAP1 that are associated with immune-related diseases, such as ankylosing spondylitis, providing human genetic validation for ERAP1's role in human disease and antigen presentation. Furthermore, in mice, genetic ablation, reduction or pharmacological inhibition of ERAP1 changes the immune repertoire, creating novel antigens with improved immunogenicity that lead to the generation of productive CD8 T cell responses and tumor growth inhibition in various syngeneic models. We have generated and profiled highly potent, species cross reactive and selective ERAP1 inhibitors through a suite of in vitro and in vivo assays to develop these as novel immunotherapy agents. In vitro CETSA measurements show potent cellular target engagement that correlates with in vitro SPR and enzyme potency. Using the model antigen SIINFEKL and Class I MHC expression as key pharmacodynamic measurements, we demonstrate Class I modulation in vitro and in vivo in line with published data from ERAP1 knockout mice. These assays are being used to elucidate the kinetics and temporal dynamics of the antigen change to optimise dosing schedules for in vivo studies. Implementation of our immunopeptidomics pipeline has demonstrated treatment with ERAP1 inhibitors both generate novel antigens in vitro and in vivo and lead to profound and consistent increased overall immunopeptidome length in mouse and human cells. To establish the functional consequence of these changes, we developed a human primary T cell immunogenicity platform. The immunogenic potential of these novel cancer associated antigens has been confirmed by the ability to stimulate IFNγ production in naïve T cells and suggests responses to these antigens could reinvigorate anti-tumor responses. Extensive assessment of mouse and human CD8 T cells responses is ongoing, in order to characterise and select Grey Wolf Therapeutics' first lead ERAP1 inhibitor for use as monotherapy or in combination with other immunotherapies such as checkpoint blockade. Citation Format: Jamie Ware, Patrick McIntyre, Kristopher Clark, Carmen Tong, Jason Shiers, Elisa Lori, Camila de Almeida, Emma Reeves, Henry Leonard, Alihussein Remtulla, Michael Ford, Nicola Ternette, Fergus Poynton, Edd James, Lesley Young, Martin Quibell, Peter I. Joyce. Potent oral ERAP1 inhibitors modify the immunopeptidome in vivo and are novel immunotherapy agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5551.

The Optimization of a Novel, Weak Bromo and Extra Terminal Domain (BET) Bromodomain Fragment Ligand to a Potent and Selective Second Bromodomain (BD2) Inhibitor.

The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors.

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

PTG-0861: A novel HDAC6-selective inhibitor as a therapeutic strategy in acute myeloid leukaemia

Dysregulated Histone Deacetylase (HDAC) activity across multiple human pathologies have highlighted this family of epigenetic enzymes as critical druggable targets, amenable to small molecule intervention. While efficacious, current approaches using non-selective HDAC inhibitors (HDACi) have been shown to cause a range of undesirable clinical toxicities. To circumvent this, recent efforts have focused on the design of highly selective HDACi as a novel therapeutic strategy. Beyond roles in regulating transcription, the unique HDAC6 (with two catalytic domains) regulates the deacetylation of α-tubulin; promoting growth factor-controlled cell motility, cell division, and metastatic hallmarks. Recent studies have linked aberrant HDAC6 function in various hematological cancers including acute myeloid leukaemia and multiple myeloma. Herein, we report the discovery, in vitro characterization, and biological evaluation of PTG-0861 (JG-265), a novel HDAC6-selective inhibitor with strong isozyme-selectivity (∼36× ) and low nanomolar potency (IC50 = 5.92 nM) against HDAC6. This selectivity profile was rationalized via in silico docking studies and also observed in cellulo through cellular target engagement. Moreover, PTG-0861 achieved relevant potency against several blood cancer cell lines (e.g. MV4-11, MM1S), whilst showing limited cytotoxicity against non-malignant cells (e.g. NHF, HUVEC) and CD-1 mice. In examining compound stability and cellular permeability, PTG-0861 revealed a promising in vitro pharmacokinetic (PK) profile. Altogether, in this study we identified a novel and potent HDAC6-selective inhibitor (∼4× more selective than current clinical standards – citarinostat, ricolinostat), which achieves cellular target engagement, efficacy in hematological cancer cells with a promising safety profile and in vitro PK.

Targeting the Water Network in Cyclin G-Associated Kinase (GAK) with 4-Anilino-quin(az)oline Inhibitors.

Water networks within kinase inhibitor design and more widely within drug discovery are generally poorly understood. The successful targeting of these networks prospectively has great promise for all facets of inhibitor design, including potency and selectivity for the target. Herein, we describe the design and testing of a targeted library of 4-anilinoquin(az)olines for use as inhibitors of cyclin G-associated kinase (GAK). GAK cellular target engagement assays, ATP binding-site modelling and extensive water mapping provide a clear route to access potent inhibitors for GAK and beyond.

Discovery and Structure-Activity Relationship Study of (Z)-5-Methylenethiazolidin-4-one Derivatives as Potent and Selective Pan-phosphatidylinositol 5-Phosphate 4-Kinase Inhibitors.

Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound 13, replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -β by the inhibitors in HEK 293T cells.

Characterization of Specific N-α-Acetyltransferase 50 (Naa50) Inhibitors Identified Using a DNA Encoded Library

Two novel compounds were identified as Naa50 binders/inhibitors using DNA-encoded technology screening. Biophysical and biochemical data as well as cocrystal structures were obtained for both compounds (3a and 4a) to understand their mechanism of action. These data were also used to rationalize the binding affinity differences observed between the two compounds and a MLGP peptide-containing substrate. Cellular target engagement experiments further confirm the Naa50 binding of 4a and demonstrate its selectivity toward related enzymes (Naa10 and Naa60). Additional analogs of inhibitor 4a were also evaluated to study the binding mode observed in the cocrystal structures.

Abstract ES12-2: Targeted DNA repair in cancer and new NUDT5 inhibitors to block hormone signalling to target breast cancer

Abstract DNA damaging agents, i.e., radio- and chemotherapy, constitute the backbone for treatment of a wide variety of cancers and may result in complete cure from the disease. Our strategy is to exploit endogenous DNA damage caused by cancer vulnerabilities, by DNA repair inhibitors using a synthetic lethal strategy. Our early work on PARP inhibitors in BRCA mutated cancers is establishing in breast cancer and here I will describe novel DNA repair targets and how these can be integrated into treatment of breast cancer. Here, I will also describe a novel link between PARP and hormone dependent cancers. Interestingly, we show that the PARP related protein NUDT5 activity is critical to elicit nuclear ATP production following hormone stimulation and a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. The NUDT5 protein is critical for 3D organoid growth of breast cancer cells. Here, we confirm the involvement of NUDT5 in ADP-ribose metabolism and identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can effectively block growth of 3D breast cancer organoids. Citation Format: T Helleday. Targeted DNA repair in cancer and new NUDT5 inhibitors to block hormone signalling to target breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES12-2.

A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

The nonreceptor protein-tyrosine phosphatase (PTP) SHP2 is encoded by the proto-oncogene PTPN11 and is a ubiquitously expressed key regulator of cell signaling, acting on a number of cellular processes and components, including the Ras/Raf/Erk, PI3K/Akt, and JAK/STAT pathways and immune checkpoint receptors. Aberrant SHP2 activity has been implicated in all phases of tumor initiation, progression, and metastasis. Gain-of-function PTPN11 mutations drive oncogenesis in several leukemias and cause developmental disorders with increased risk of malignancy such as Noonan syndrome. Until recently, small molecule-based targeting of SHP2 was hampered by the failure of orthosteric active-site inhibitors to achieve selectivity and potency within a useful therapeutic window. However, new SHP2 allosteric inhibitors with excellent potency and selectivity have sparked renewed interest in the selective targeting of SHP2 and other PTP family members. Crucially, drug discovery campaigns focusing on SHP2 would greatly benefit from the ability to validate the cellular target engagement of candidate inhibitors. Here, we report a cellular thermal shift assay that reliably detects target engagement of SHP2 inhibitors. Using this assay, based on the DiscoverX InCell Pulse enzyme complementation technology, we characterized the binding of several SHP2 allosteric inhibitors in intact cells. Moreover, we demonstrate the robustness and reliability of a 384-well miniaturized version of the assay for the screening of SHP2 inhibitors targeting either WT SHP2 or its oncogenic E76K variant. Finally, we provide an example of the assay's ability to identify and characterize novel compounds with specific cellular potency for either WT or mutant SHP2.

Cellular Target Engagement Approaches to Monitor Epigenetic Reader Domain Interactions.

Malfunctions in the basic epigenetic mechanisms such as histone modifications, DNA methylation, and chromatin remodeling are implicated in a number of cancers and immunological and neurodegenerative conditions. Within GlaxoSmithKline (GSK) we have utilized a number of variations of the NanoBRET technology for the direct measurement of compound-target engagement within native cellular environments to drive high-throughput, routine structure-activity relationship (SAR) profiling across differing epigenetic targets. NanoBRET is a variation of the bioluminescence resonance energy transfer (BRET) methodology utilizing proteins of interest fused to either NanoLuc, a small, high-emission-intensity luciferase, or HaloTag, a modified dehalogenase enzyme that can be selectively labeled with a fluorophore. The combination of these two technologies has enabled the application of NanoBRET to biological systems such as epigenetic protein-protein interactions, which have previously been challenging. By synergizing target engagement assays with more complex primary cell phenotypic assays, we have been able to demonstrate compound-target selectivity profiles to enhance cellular potency and offset potential liability risks. Additionally, we have shown that in the absence of a robust, cell phenotypic assay, it is possible to utilize NanoBRET target engagement assays to aid chemistry in progressing at a higher scale than would have otherwise been achievable. The NanoBRET target engagement assays utilized have further shown an excellent correlation with more reductionist biochemical and biophysical assay systems, clearly demonstrating the possibility of using such assay systems at scale, in tandem with, or in preference to, lower-throughput cell phenotypic approaches.

Abstract B126: A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo

Abstract METTL3 is an RNA methyltransferase which is responsible for the deposition of N-6-methyladenosine (m6A) on mRNA targets such as SP1, to modulate their stability and expression. METTL3 was identified as an essential gene for the growth of AML cells and proposed as a novel target for cancer therapy (Barbieri 2017). We present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3, which recapitulate the genetic validation of METTL3 as a novel cancer target using a pharmacological audit trail. Small molecule inhibitors from 2 distinct chemical series were identified and optimised using a structure-guided medicinal chemistry platform. Compounds 1 and 2 are from different series, and both showed biochemical inhibition of METTL3 enzyme with single digit nanomolar potency. Direct binding to METTL3 was confirmed by SPR with comparable potency. Compound 3 is an inactive analog which was confirmed inactive in the enzyme assay (IC50 >50microM). Compounds 1-2 are selective for METTL3 and did not inhibit a panel of other RNA, DNA or protein methyltransferases tested (>10microM IC50). Cellular target engagement was confirmed by the demonstration that compounds 1 and 2 inhibited SP1 and Brd4 protein expression with submicromolar potency, whereas the inactive analog compound 3 had no effect. Compounds 1 and 2 treatment of MOLM13 cells inhibited their proliferation which correlated with SP1 inhibition, and compound 3 had no effect, demonstrating that their activity was METTL3-dependent. Compound 1 has excellent oral bioavailability with good dose-proportional exposure in mice and a half-life of 3.5 hrs, and was well-tolerated with no body weight loss or clinical signs. Compound 1 was evaluated for anti-tumor effects in an MLL-AF9 driven primary murine AML model. 30 mg/kg daily oral dosing of compound 1 gave a significant reduction in AML expansion and a reduction in spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by reduction of METTL3-dependent m6A targets. We have described the comprehensive characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrated their activity and utility using biochemical, cellular and in vivo systems. We have demonstrated that inhibition of METTL3 by small molecules in vivo leads to a pronounced anti-tumor effect in a physiologically relevant model of acute myeloid leukemia. To our knowledge, this is the first demonstration of in vivo activity of inhibitors of an RNA methyltransferase and providing proof of concept that RNA modifying enzymes are a new target class for the development of novel cancer therapeutics. Citation Format: Mark Albertella, Wesley Blackaby, Richard Fosbeary, Alan Hendrick, Dan Leggate, Yaara Ofir-Rosenfeld, Alexandra Sapetschnig, Konstantinos Tzelepis, Eliza Yankova, Tony Kouzarides, Oliver Rausch. A small molecule inhibitor of the RNA m6A writer METTL3 inhibits the development of acute myeloid leukemia (AML) in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B126. doi:10.1158/1535-7163.TARG-19-B126

Pharmacological Inhibition of the RNA m6a Writer METTL3 As a Novel Therapeutic Strategy for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. New evidence has revealed an important role for RNA modifications in cancer development and maintenance via the catalytic function of RNA-modifying enzymes. We and others have recently shown that METTL3, the RNA methyltransferase responsible for the deposition of N-6-methyl groups on adenosine (m6A) in mRNA, is a promising therapeutic target for AML1,2. Here we present the in vitro and in vivo characterization of novel small molecule inhibitors of METTL3 as an effective therapeutic strategy in AML. Recently, we generated a comprehensive catalogue of RNA-modifying enzymes that are essential for AML cells using CRISPR-Cas9 recessive screens and characterised METTL3 as a novel therapeutic candidate through its effects on mRNA translational efficiency of key leukemia oncogenes1. Using a structure-guided medicinal chemistry platform we developed and optimised small molecule inhibitors of METTL3 from 2 distinct chemical series. Here we demonstrate that compounds 1 and 2 show biochemical inhibition of METTL3 enzyme with single digit nanomolar potency, while direct binding to METTL3 was confirmed by Surface Plasmon Resonance (SPR) analysis with comparable potency between compounds. Additionally, we developed compound 3 as an inactive analog which was confirmed inactive in enzyme assays (>50 µM IC50). Importantly, we verified that compounds 1 and 2 are selective for METTL3 and do not inhibit a panel of other RNA, DNA or protein methyltransferases tested (>10 µM IC50). Cellular target engagement was confirmed by demonstrating that compounds 1 and 2 reduced m6A levels and inhibited the protein expression of METTL3-dependent m6A substrates in mouse and human AML models, including SP1, with nanomolar potency. Furthermore, treatment of MOLM13 cells with compounds 1 and 2 inhibited their proliferation with comparable potency to SP1 inhibition. The same anti-proliferative effect was observed using a large panel of human AML cell lines. In addition, polyribosome profiling in MOLM13 cells treated with compounds 1 and 2 revealed enhanced blocking of mRNA translation, mirroring the effects derived from the genetic inhibition of METTL3. Notably, all of the above effects were not observed when the inactive analog (compound 3) was used, further highlighting the specificity and sensitivity of our active candidates. We subsequently performed in vivo characterisation of compound 1. This compound exhibited excellent bioavailability after oral or intraperitoneal administration with good dose-proportional exposure in mice and a half-life of 3.5 hours. It also appeared to be well-tolerated with no body weight loss or clinical signs of toxicity. We also evaluated its anti-tumor effects in patient derived xenotransplantation experiments (PDX) as well as transplantation experiments using an MLL-AF9 driven primary murine AML model. Daily dosing of 30 mg/kg significantly inhibited AML expansion and reduced spleen weight compared to vehicle control, indicating a pronounced anti-tumor effect in vivo. Target engagement was confirmed in bone marrow and spleen as measured by the reduction of METTL3-dependent m6A targets. Importantly, we went on to demonstrate that, while the pharmacological inhibition of METTL3 is required for AML cell survival, it was dispensable for normal hematopoiesis. Collectively, we describe the detailed characterization of potent and selective inhibitors of the METTL3 RNA methyltransferase, and demonstrate their activity and utility using biochemical, cellular and in vivo systems. We show that inhibition of METTL3 by small molecules in vivo leads to strong anti-tumor effects in physiologically and clinically relevant models of AML. To our knowledge, this is the first study demonstrating in vivo activity of inhibitors of an RNA methyltransferase, hence providing proof of concept that RNA modifying enzymes represent a new target class for anti-cancer therapeutics. References Barbieri, I. et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature552, 126-131, doi:10.1038/nature24678 (2017). Vu, L. P. et al. The N(6)-methyladenosine (m(6)A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells. Nat Med23, 1369-1376, doi:10.1038/nm.4416 (2017). Disclosures Yankova: STORM THERAPEUTICS: Employment. Fosbeary:STORM THERAPEUTICS: Employment. Hendrick:STORM THERAPEUTICS: Employment. Leggate:STORM THERAPEUTICS: Employment. Ofir-Rosenfeld:STORM THERAPEUTICS: Employment. Sapetschnig:STORM THERAPEUTICS: Employment. Albertella:STORM THERAPEUTICS: Employment. Blackaby:STORM THERAPEUTICS: Employment. Rausch:STORM THERAPEUTICS: Employment. Vassiliou:Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Kouzarides:STORM THERAPEUTICS: Equity Ownership.

Perspective on CETSA Literature: Toward More Quantitative Data Interpretation.

The cellular thermal shift assay (CETSA) was introduced in 2013 to investigate drug-target engagement inside live cells and tissues. As with all thermal shift assays, the response measured by CETSA is not simply governed by ligand affinity to the investigated target protein, but the thermodynamics and kinetics of ligand binding and protein unfolding also contribute to the observed protein stabilization. This limitation is commonly neglected in current applications of the method to validate the target of small-molecule probes. Instead, there is an eagerness to make direct comparisons of CETSA measurements with functional and phenotypic readouts from cells at 37 °C. Here, we present a perspective of the early CETSA literature and put the accumulated data into a quantitative context. The analysis includes annotation of ~270 peer-reviewed papers, the majority of which do not consider the underlying biophysical basis of CETSA. We also detail what future technology developments are needed to enable CETSA-based optimization of structure-activity relationships and more appropriate comparisons of these data with functional or phenotypic responses. Finally, we describe ongoing developments in assay formats that allow for CETSA measurements at single-cell resolution, with the aspiration to allow differentiation in cellular target engagement between cells in co-cultures and more complex models, such as organoids and potentially even tissue.

Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Abstract 324: BRAF/MEK inhibitors selectively prime melanoma for Mcl-1 dependence and sensitize to Mcl-1 inhibition

Abstract Frontline treatment for patients diagnosed with metastatic melanoma harboring a V600 BRAF mutation is a combination of BRAF/MEK inhibitors. Despite advances in targeted therapies used to treat melanoma, the majority of patients will relapse due to acquired resistance. The intrinsic apoptotic pathway is frequently dysregulated in cancer, promoting cell survival and resistance mechanisms. The anti-apoptotic Mcl-1 protein was identified as a key resistance factor to vemurafenib, a mutant BRAF inhibitor, and likely plays a role in preventing apoptosis in the context of dual BRAF/MEK inhibitor resistance. Activation of apoptosis by using small molecule inhibitors of the anti-apoptotic Bcl-2 proteins has become a validated therapeutic approach and can re-sensitize cancers to standard of care treatments. We found that melanoma cell lines display a heterogeneous dependency on anti-apoptotic proteins. In this study, we systematically investigated the role of Mcl-1 in vemurafenib resistance in melanoma cell lines using genetic and pharmacological inhibition of Mcl-1. Using the functional BH3 profiling assay, we discovered that melanoma cell lines resistant to vemurafenib are increasingly primed for Mcl-1 dependence. Additionally, applying dynamic BH3 profiling we demonstrated that melanoma cell lines subjected to short term treatment with vemurafenib showed greater dependency upon Mcl-1 for survival, thus serving as a selective priming agent. Consistent with these findings, both vemurafenib resistant and short-term vemurafenib pre-treated melanoma cell lines displayed increased sensitivity to Mcl-1 genetic silencing using siRNA and direct Mcl-1 inhibition using small-molecule inhibitors developed by our group. We further demonstrate significant synergy in the induction of apoptosis induced by the combination of vemurafenib and Mcl-1 inhibitors such as UM-101. In a similar way, Mcl-1 priming and increased sensitivity to Mcl-1 inhibitors was shown in melanoma cells pre-treated with MEK inhibitors. We further studied the mechanism of apoptosis induced by Mcl-1 inhibitors and validated their cellular target engagement. These studies provide evidence that targeting Mcl-1 represents a promising therapeutic strategy for treating patients with resistant melanomas and aids in the design of rational combination therapies before resistance is acquired. Citation Format: Karson J. Kump, Lei Miao, Nurul Ansari, David B. Lombard, Zaneta Nikolovska-Coleska. BRAF/MEK inhibitors selectively prime melanoma for Mcl-1 dependence and sensitize to Mcl-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 324.

Abstract 1327: UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia

Abstract Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I ‘gatekeeper’ mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib. Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML. Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity. Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML. Citation Format: Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1327.

Abstract 3588: Identification of a potent, orally bioavailable and selective MCT4 Inhibitor for the treatment of solid Warburg tumors

Abstract A key metabolic alteration in tumor cells is an elevated glycolysis rate, resulting in the production of increased amounts of protons and lactate leading to lactic acidosis within the tumor microenvironment. The tumor-promoting effects of lactic acidosis are versatile, ranging from stimulation of proliferation, angiogenesis and metastasis to the reshaping of the tumor immune environment to a tumor-permissive phenotype. In tumors lactate is transported across the plasma membrane by the monocarboxylate transporters 1 and 4 (MCT 1/4) in symport with a proton. While MCT1 is expressed almost ubiquitously at low levels, MCT4 expression is regulated by hypoxia-inducible factor 1α (Hif1α), the central player in the cellular response to hypoxia. Thus, MCT4 is present in hypoxic, highly glycolytic cells. Given their central role in prevention of intracellular acidification and lactate transport, MCTs appear to be indispensable for glycolytic tumor growth. Since MCT4 has the strongest implication on the development and aggressiveness of diverse cancer types it has emerged as a new target for the treatment of solid tumors. We have identified MCT4 specific inhibitors structurally based on an internal compound library using cell-based lactate export assays. A subsequent extensive medicinal chemistry program was able to significantly improve activity and selectivity of the primary structures. Cellular target engagement was verified by cell-based assays like cellular thermal shift assay (CETSA). These efforts resulted in the identification of highly potent, orally bioavailable and selective MCT4 inhibitors (MCT4 IC50 < 20nM; MCT1 IC50 > 100µM) with good physicochemical properties and beneficial pharmacokinetics in mouse. In vivo efficacy and tumor lactate modulation were evaluated in NCI-H358 xenograft and MC38 syngeneic mouse models. Thereby we explore the potential of our MCT4 inhibitor to act as a single anti-cancer agent or to synergize with other established therapies like angiogenesis inhibitors or cancer immunotherapeutics. Citation Format: Nina Hambruch, Barbara Herkert, Christian Gege, Aurelie Mallinger, Johannes Fabian, Olaf Kinzel, Yansong Wang, Gero Fink, Michael Albers, Anette Funk, Annika Bittman-Waetzig, Tilly Fleckenstein, Floriane Braun, Claus Kremoser. Identification of a potent, orally bioavailable and selective MCT4 Inhibitor for the treatment of solid Warburg tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3588.

Development of Highly Potent, Selective, and Cellular Active Triazolo[1,5- a]pyrimidine-Based Inhibitors Targeting the DCN1-UBC12 Protein-Protein Interaction.

The cullin-RING ubiquitin ligases (CRLs) are responsible for about 20% of cellular protein degradation and regulate diverse cellular processes, and the dysfunction of CRLs is implicated in human diseases. Targeting the CRLs has become an emerging strategy for the treatment of human diseases. Herein, we describe the discovery of a hit compound from our in-house library and further structure-based optimizations, which have enabled the identification of new triazolo[1,5- a]pyrimidine-based inhibitors targeting the DCN1-UBC12 interaction. Compound WS-383 blocks the DCN1-UBC12 interaction (IC50 = 11 nM) reversibly and shows selectivity over selected kinases. WS-383 exhibits cellular target engagement to DCN1 in MGC-803 cells. WS-383 inhibits Cul3/1 neddylation selectively over other cullins and also induces accumulation of p21, p27, and NRF2. Collectively, targeting the DCN1-UBC12 interaction would be a viable strategy for selective neddylation inhibition of Cul3/1 and may be of therapeutic potential for disease treatment in which Cul3/1 is dysregulated.

Synthesis, structure-activity relationship studies and biological characterization of new [1,2,4]triazolo[1,5-a]pyrimidine-based LSD1/KDM1A inhibitors

The histone lysine specific demethylase 1 (LSD1/KDM1A) is implicated in the development of cancers, targeting LSD1 has been recognized as a promising strategy for cancer therapy. To date, some small-molecule inhibitors are currently being investigated in clinical trials. Herein we report the design, synthesis and biochemical characterization of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new LSD1 inhibitors. Of these compounds, compound C26 inhibited LSD1 in a reversible manner (IC50 = 1.72 μM) and showed selectivity to LSD1 over MAO-A/B. Besides, compound C26 displayed FAD-competitive binding to LSD1. Interestingly, C26 did not inhibit horseradish peroxidase (HRP) and quench H2O2, thus excluding the possibility that LSD1 inhibition by C26 was due to the HRP inhibition and consumption of H2O2. In LSD1 overexpressed A549 cells, compound C26 concentration-dependently induced accumulation of H3K4me1/me2 and H3K9me2 and showed cellular target engagement to LSD1. Additionally, compound C26 significantly inhibited migration of A549 cells in a concentration-dependent manner, further western blot analysis showed that C26 increased expression levels of epithelial cell markers E-Cadherin and Claudin-1, down-regulated mesenchymal cell marker N-Cadherin and the upstream transcription factors Snail and Slug. Docking studies were also performed to rationalize the potency of C26 toward LSD1. To conclude, the [1,2,4]triazolo[1,5-a]pyrimidine could serve as a promising scaffold for the development of new LSD1 inhibitors.