The allosteric inhibitor ABL001 enables dual targeting of BCR-ABL1.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment.

Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS­ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS­ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

The R882H DNMT3A hot spot mutation stabilizes the formation of large DNMT3A oligomers with low DNA methyltransferase activity.

DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.

A conserved salt bridge in the G loop of multiple protein kinases is important for catalysis and for in vivo Lyn function.

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors.

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr-Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr-Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

Trisomy 12 and elevated GLI1 and PTCH1 transcript levels are biomarkers for Hedgehog-inhibitor responsiveness in CLL.

Hedgehog (HH) signaling is activated in various lymphoid malignancies, but conflicting results exist about its role in chronic lymphocytic leukemia (CLL). Here, we demonstrate that the expression of essential HH pathway components like GLI1, PTCH1, and the HH ligands is highly diverse in CLL. A subset of 36.7% of 60 tested CLL samples responded to all 3 SMOOTHENED (SMO) inhibitors, whereas 40% were completely resistant. Responsiveness correlated with elevated GLI1 and PTCH1 transcript levels and the presence of trisomy 12, whereas no other karyotype correlated with responsiveness. All trisomy 12 CLLs displayed constitutive HH pathway activation driven by autocrine DESERT HH (DHH) ligand secretion, which could be blocked by the HH-blocking Ab 5E1. Cocultures with DHH-expressing BM stromal cells reduced sensitivity of CLLs to SMO-inhibitor treatment by activation of noncanonical ERK phosphorylation directly downstream of the PTCH1 receptor without involvement of SMO and could be overcome by the HH-blocking Ab 5E1 or a combination of SMO and ERK inhibitors. Our results demonstrate that the HH-signaling pathway is an interesting therapeutic target for a subset of patients with CLL, characterized by high GLI1 and PTCH1 transcript levels, and all patients with trisomy 12 and indicate HH-blocking Abs to be favorable over SMO inhibitors in overcoming stroma-mediated protective effects.

Essential role of stromally induced hedgehog signaling in B-cell malignancies.

Interaction of cancer cells with their microenvironment generated by stromal cells is essential for tumor cell survival and influences the localization of tumor growth. Here we demonstrate that hedgehog ligands secreted by bone-marrow, nodal and splenic stromal cells function as survival factors for malignant lymphoma and plasmacytoma cells derived from transgenic Emu-Myc mice or isolated from humans with these malignancies. Hedgehog pathway inhibition in lymphomas induced apoptosis through downregulation of Bcl2, but was independent of p53 or Bmi1 expression. Blockage of hedgehog signaling in vivo inhibited expansion of mouse lymphoma cells in a syngeneic mouse model and reduced tumor mass in mice with fully developed disease. Our data indicate that stromally induced hedgehog signaling may provide an important survival signal for B- and plasma-cell malignancies in vitro and in vivo. Disruption of this interaction by hedgehog pathway inhibition could provide a new strategy in lymphoma and multiple myeloma therapy.

Maintenance of adenomatous polyposis coli (APC)-mutant colorectal cancer is dependent on Wnt/beta-catenin signaling.

Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible ß-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/ß-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon ß-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/ß-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear ß-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/ß-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/ß-catenin pathway.

Requirement of Src kinases Lyn, Hck and Fgr for BCR-ABL1-induced B-lymphoblastic leukemia but not chronic myeloid leukemia.

The Abl kinase inhibitor imatinib mesylate is the preferred treatment for Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML) in chronic phase but is much less effective in CML blast crisis or Ph(+) B-cell acute lymphoblastic leukemia (B-ALL). Here, we show that Bcr-Abl activated the Src kinases Lyn, Hck and Fgr in B-lymphoid cells. BCR-ABL1 retrovirus-transduced marrow from mice lacking all three Src kinases efficiently induced CML but not B-ALL in recipients. The kinase inhibitor CGP76030 impaired the proliferation of B-lymphoid cells expressing Bcr-Abl in vitro and prolonged survival of mice with B-ALL but not CML. The combination of CGP76030 and imatinib was superior to imatinib alone in this regard. The biochemical target of CGP76030 in leukemia cells was Src kinases, not Bcr-Abl. These results implicate Src family kinases as therapeutic targets in Ph(+) B-ALL and suggest that simultaneous inhibition of Src and Bcr-Abl kinases may benefit individuals with Ph(+) acute leukemia.

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site.

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr-Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

Nonclinical pharmacokinetics and in vitro metabolism of H3B-6545, a novel selective ERα covalent antagonist (SERCA).

PURPOSE: H3B-6545, a novel selective estrogen receptor (ER)α covalent antagonist (SERCA) which inactivates both wild-type and mutant ERα, is in clinical development for the treatment of metastatic breast cancer. Preclinical studies were conducted to characterize the pharmacokinetics and metabolism of H3B-6545 in rat and monkeys. METHODS: The clearance and metabolic profiles of H3B-6545 were studied using rat, monkey and human hepatocytes, and reaction phenotyping was done using recombinant human cytochrome P450 enzymes. Blood stability, protein binding, and permeability were also determined in vitro. Pharmacokinetics of H3B-6545 was assessed after both intravenous and oral dosing. A nonclinical PBPK model was developed to assess in vitro-in vivo correlation of clearance. RESULTS: H3B-6545 had a terminal elimination half-life of 2.4 h in rats and 4.0 h in monkeys and showed low to moderate bioavailability, in line with the in vitro permeability assessment. Plasma protein binding was similar across species, at 99.5-99.8%. Nine metabolites of H3B-6545 were identified in hepatocyte incubations, none of which were unique to humans. Formation of glutathione-related conjugate of H3B-6545 was minimal in vitro. H3B-6545, a CYP3A substrate, is expected to be mostly cleared via hepatic phase 1 metabolism. Hepatocyte clearance values were used to adequately model the time-concentration profiles in rat and monkey. CONCLUSIONS: We report on the absorption and metabolic fate and disposition of H3B-6545 in rats and dogs and illustrate that in vitro-in vivo correlation of clearance is possible for targeted covalent inhibitors, provided reactivity is not a predominant mechanism of clearance.

Effect of a high-fat meal on the relative bioavailability of H3B-6527, a novel FGFR4 inhibitor, in healthy volunteers.

PURPOSE: This Phase I study estimated the effect of a high-fat meal on the pharmacokinetics (PK) of H3B-6527, a covalent inhibitor of the fibroblast growth factor receptor (FGFR) 4 in clinical development for hepatocellular carcinoma and intrahepatic cholangiocarcinoma. METHODS: In this randomized, single center, single-dose, open-label, 2-period crossover study 12 healthy male volunteers, aged 18-55 years old, received a single 200-mg dose of H3B-6527 (capsule) following an overnight fast or a high-fat breakfast. PK samples were collected serially up to 36 h postdose. H3B-6527 concentrations were measured using a validated high-performance liquid chromatography tandem mass spectrometry method. PK data were analyzed using a noncompartmental approach based on a mixed-effects model. The safety and tolerability of H3B-6527 were also assessed. RESULTS: H3B-6527 plasma exposure increased after a high-fat meal with fed/fasted ratios of the geometric means (90% confidence interval) of 174% (102-298%) for Cmax and 246% (146-415%) for AUC0-t. Food delayed and prolonged absorption of H3B-6527, with a fed/fasted ratio for tmax of 200% (137-263%). PK variability was lower under the fed condition, as illustrated by the CV% for Cmax and AUC0-t of 41.9-54.5% (fed) versus 64.3-70.4% (fasted). CONCLUSIONS: A single 200 mg dose of H3B-6527 was safe and generally well tolerated when administered to healthy adult males. A high-fat meal significantly increased exposure to H3B-6527, from 1.5- to 2.5-fold in the systemic circulation, compared to administration under fasted conditions. Food delayed and prolonged absorption of H3B-6527. In general, lower inter-subject variability was observed in the fed state in healthy volunteers. TRIAL REGISTRATION: ClinicalTrials.gov.: NCT03424577.

Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators.

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase.

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.

H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers.

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function. Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function, but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers.

Discovery of Asciminib (ABL001), an Allosteric Inhibitor of the Tyrosine Kinase Activity of BCR-ABL1.

Chronic myelogenous leukemia (CML) arises from the constitutive activity of the BCR-ABL1 oncoprotein. Tyrosine kinase inhibitors (TKIs) that target the ATP-binding site have transformed CML into a chronic manageable disease. However, some patients develop drug resistance due to ATP-site mutations impeding drug binding. We describe the discovery of asciminib (ABL001), the first allosteric BCR-ABL1 inhibitor to reach the clinic. Asciminib binds to the myristate pocket of BCR-ABL1 and maintains activity against TKI-resistant ATP-site mutations. Although resistance can emerge due to myristate-site mutations, these are sensitive to ATP-competitive inhibitors so that combinations of asciminib with ATP-competitive TKIs suppress the emergence of resistance. Fragment-based screening using NMR and X-ray yielded ligands for the myristate pocket. An NMR-based conformational assay guided the transformation of these inactive ligands into ABL1 inhibitors. Further structure-based optimization for potency, physicochemical, pharmacokinetic, and drug-like properties, culminated in asciminib, which is currently undergoing clinical studies in CML patients.

Discovery of Selective Estrogen Receptor Covalent Antagonists for the Treatment of ERαWT and ERαMUT Breast Cancer.

Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

B-Raf kinase inhibitors for cancer treatment.

The Raf-MEK-ERK signaling pathway is critical for cell survival, growth, proliferation and tumorigenesis. Among the three isoforms of Raf protein kinases, in vitro and in vivo studies have shown that B-Raf functions as the primary MEK activator. B-Raf is one of the most frequently mutated genes in human cancers with a high prevalence in melanoma, and many of the B-Raf mutations activate the kinase activity of B-Raf. B-Raf kinase represents an excellent target for anticancer therapy based on preclinical target validation, epidemiology and drugability. Several small-molecule inhibitors of B-Raf kinase are currently undergoing clinical evaluation, with others due to enter clinical development in the near future.

A general strategy for creating "inactive-conformation" abl inhibitors.

Kinase inhibitors that bind to the ATP cleft can be broadly classified into two groups: those that bind exclusively to the ATP site with the kinase assuming a conformation otherwise conducive to phosphotransfer (type I), and those that exploit a hydrophobic site immediately adjacent to the ATP pocket made accessible by a conformational rearrangement of the activation loop (type II). To date, all type II inhibitors were discovered by using structure-activity-guided optimization strategies. Here, we describe a general pharmacophore model of type II inhibition that enables a rational "hybrid-design" approach whereby a 3-trifluoromethylbenzamide functionality is appended to four distinct type I scaffolds in order to convert them into their corresponding type II counterparts. We demonstrate that the designed compounds function as type II inhibitors by using biochemical and cellular kinase assays and by cocrystallography with Abl.