Abl Kinase Inhibitor Research Articles

Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.

Focal adhesion kinase family is involved in matrix contraction by transdifferentiated Müller cells

Transdifferentiated Müller cells that adopt a fibroblastic/myofibroblastic phenotype have been identified in epiretinal membranes (ERMs) in several ocular disorders, and have been implicated to play a role in the formation and/or the contraction of ERMs. We have previously demonstrated that dasatinib, a dual inhibitor of Src-family kinases and Abl kinase, can prevent matrix contraction by transdifferentiated Müller cells. In this study, we examined molecules involved in matrix contraction downstream of primary dasatinib targets. Tyrosine phosphorylation of focal adhesion kinase (FAK) family members FAK and PYK2 was significantly reduced by dasatinib, and select inhibitors for these kinases PF431396, which inhibits both FAK and PYK2, and PF573228, which only inhibits FAK and not PYK2, significantly reduced matrix contraction by transdifferentiated Müller cells. Dasatinib and PF431396 significantly reduced phosphorylation of Hic-5, a protein implicated to play a role in focal adhesions and cell signaling. Our data shows that FAK family members are involved in matrix contraction by transdifferentiated Müller cells, and also implicates that Hic-5 is situated downstream of the FAK family within the signaling pathway.

Anti-leukemia activity of a Hsp70 inhibitor and its hybrid molecules

In this study we examined the anti-leukemia activity of a small molecule inhibitor of Hsp70 proteins, apoptozole (Az), and hybrids in which it is linked to an inhibitor of either Hsp90 (geldanamycin) or Abl kinase (imatinib). The results of NMR studies revealed that Az associates with an ATPase domain of Hsc70 and thus blocks ATP binding to the protein. Observations made in the cell study indicated that Az treatment promotes leukemia cell death by activating caspase-dependent apoptosis without affecting the caspase-independent apoptotic pathway. Importantly, the hybrids composed of Az and geldanamycin, which have high inhibitory activities towards both Hsp70 and Hsp90, exhibit enhanced anti-leukemia activity relative to the individual inhibitors. However, the Az and imatinib hybrids have weak inhibitory activities towards Hsp70 and Abl, and display lower cytotoxicity against leukemia cells compared to those of the individual constituents. The results of a mechanistic study showed that the active hybrid molecules promote leukemia cell death through a caspase-dependent apoptotic pathway. Taken together, the findings suggest that Hsp70 inhibitors as well as their hybrids can serve as potential anti-leukemia agents.

Computational analysis of ABL kinase mutations allows predicting drug sensitivity against selective kinase inhibitors

The ABL kinase inhibitor imatinib has been used as front-line therapy for Philadelphia-positive chronic myeloid leukemia. However, a significant proportion of imatinib-treated patients relapse due to occurrence of mutations in the ABL kinase domain. Although inhibitor sensitivity for a set of mutations was reported, the role of less frequent ABL kinase mutations in drug sensitivity/resistance is not known. Moreover, recent reports indicate distinct resistance profiles for second-generation ABL inhibitors. We thus employed a computational approach to predict drug sensitivity of 234 point mutations that were reported in chronic myeloid leukemia patients. Initial validation analysis of our approach using a panel of previously studied frequent mutations indicated that the computational data generated in this study correlated well with the published experimental/clinical data. In addition, we present drug sensitivity profiles for remaining point mutations by computational docking analysis using imatinib as well as next generation ABL inhibitors nilotinib, dasatinib, bosutinib, axitinib, and ponatinib. Our results indicate distinct drug sensitivity profiles for ABL mutants toward kinase inhibitors. In addition, drug sensitivity profiles of a set of compound mutations in ABL kinase were also presented in this study. Thus, our large scale computational study provides comprehensive sensitivity/resistance profiles of ABL mutations toward specific kinase inhibitors.

Allosteric Plus Catalytic BCR–ABL1 Inhibitors Promote Durable Responses

Abstract A selective allosteric ABL1 kinase inhibitor suppresses the growth of BCR–ABL1-driven CML.

19F-NMR-Based Dual-Site Reporter Assay for the Discovery and Distinction of Catalytic and Allosteric Kinase Inhibitors.

In modern kinase drug discovery, allosteric inhibitors have become a focus of attention due to their potential selectivity, but such compounds are difficult to identify. Here we describe an NMR-based competition assay using 19F-containing reporter molecules, which allows for rapid identification and discrimination between ATP-competitive and allosteric kinase inhibitors. We illustrate the principle of such a dual-site competition assay with the example of catalytic and allosteric ABL1 kinase inhibitors. The assay can also be used to identify and characterize mixed binding modes of well-known drugs, as shown for crizotinib and fingolimod.

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.

Pepsi® or Coke®? Influence of acid on dasatinib absorption.

Dasatinib is a second generation ABL kinase inhibitor used in the management of chronic myeloid leukemia or Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). Dasatinib's bioavailability is highly dependent on gastric pH. When proton-pump inhibitors (PPIs) are co-administered with dasatinib, absorption is significantly reduced. Cola intake at the time of drug administration has been demonstrated to lead to relevant increases in the bioavailability for other acid labile drugs during PPI treatment. This manuscript reviews the relevant literature supporting a strategy of temporarily lowering the gastric pH with a carbonated beverage at the time of drug administration. The use of cola provides an easy to implement way to significantly improve dasatinib bioavailability, especially during concomitant use of a PPI.

Development of a Predictive Pharmacophore Model and a 3D-QSAR Study for an in silico Screening of New Potent Bcr-Abl Kinase Inhibitors.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder, characterized, in most cases, by the presence of the Bcr-Abl fusion oncogene. Bcr-Abl is a constitutively active tyrosine kinase that is responsible for the malignant transformation. Targeting the Bcr-Abl kinase is an attractive treatment strategy for CML. First and second generation Bcr-Abl inhibitors have focused on targeting the ATP-binding domain of the kinase. Mutations in that region are relatively resistant to drug manipulation. Therefore, non-ATP-competitive agents have been recently developed and tested. In the present study, in an attempt to aid the design of new chemotypes with enhanced cytotoxicity against K562 cells, 3D pharmacophore models were generated and 3D-QSAR CoMFA and CoMSIA studies were carried out on the 33 novel Abl kinase inhibitors (E)-α-benzylthio chalcones synthesized by Reddy et al. A five-point pharmacophore with a hydrogen bond acceptor, two hydrophobic groups and two aromatic rings as pharmacophore features, and a statistically significant 3D-QSAR model with excellent predictive power were developed. The pharmacophore model was also used for alignment of the 33 compounds in a CoMFA/CoMSIA analysis. The contour maps of the fields of CoMFA and CoMSIA models were utilized to provide structural insight into how these molecules promote their toxicity. The possibility of using this model for the design of drugs for the treatment of β-thalassemia and sickle cell disease (SCD), since several Bcr-Abl inhibitors are able to promote erythroid differentiation and γ-globin expression in CML cell lines and primary erythroid cells is discussed.

Combining the Allosteric ABL1 Tyrosine Kinase Inhibitor ABL001 with ATP-Competitive Inhibitors to Suppress Resistance in Chronic Myeloid Leukemia

The successful design and application of ABL1 tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia (CML) has become a paradigm for molecularly targeted cancer therapies. However, resistance to therapy can develop and is most commonly attributed to acquired mutations within the kinase domain of the causative BCR-ABL1 oncoprotein. While the set of ABL1 kinase inhibitors currently available in the clinic facilitates management of many of these resistant BCR-ABL1 mutations, all clinically approved ABL1 kinase inhibitors for CML to date bind to the catalytic site within the kinase in an ATP-competitive manner. An alternative strategy to circumventing mutations in this region which has received recent attention involves the targeting of allosteric sites on BCR-ABL1 kinase. Here, we evaluated ABL001, a small-molecule inhibitor which binds highly selectively to the myristoyl pocket on ABL1 kinase, for its capacity to inhibit known imatinib-resistant BCR-ABL1 single and compound mutations. ABL001 potently inhibited the proliferation of Ba/F3 cells expressing native BCR-ABL1 or a variety of BCR-ABL1 point mutations (IC50 range: 1.3-113.5 nM), with no observed toxicity to parental Ba/F3 cells up to 10 μM. Interestingly, however, ABL001 demonstrated little to no activity against a small panel of BCR-ABL1 compound mutations tested (G250E/T315I, E255K/T315I, E255V/T315I). Accelerated mutagenesis screens for ABL001 revealed dose-dependent reduction in the emergence of resistant clones, as well as novel BCR-ABL1 mutations at a subset of kinase domain and C-terminal residues lining the myristoyl pocket which are resistant to ABL001 but remain sensitive to ATP-competitive ABL1 kinase inhibitors. Intriguingly, similar mutagenesis screens conducted using ABL001 in combination with relevant ATP-competitive ABL1 kinase inhibitors showed marked suppression of resistant outgrowth compared to either drug alone, both in the setting of single and compound mutations. Furthermore, ABL001 demonstrated dose-dependent inhibition of myeloid colony formation of human CML cells, and this efficacy was further enhanced in the context of co-treatment with nilotinib or ponatinib. Together, these findings suggest that use of an allosteric ABL1 inhibitor such as ABL001 in combination with other currently approved ATP-competitive inhibitors may offer a meaningful strategy to further mitigate the emergence of resistance in CML patients both in a frontline and second-line treatment capacity. DisclosuresHeinrich:MolecularMD: Consultancy, Equity Ownership; ARIAD: Consultancy, Research Funding; Blueprint Medicines: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Onyx: Consultancy; Pfizer: Consultancy; Bayer: Research Funding; BMS: Research Funding. Druker:Roche: Consultancy; Lorus: Consultancy, Equity Ownership; AstraZeneca: Consultancy; Pfizer: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; CTI: Consultancy, Equity Ownership; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; Curis: Patents & Royalties; D3 Oncology Solutions: Consultancy; Agios: Honoraria; Ambit BioSciences: Consultancy; Pfizer: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; Cylene: Consultancy, Equity Ownership; BMS: Research Funding; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute.

Characterization of the Genomic Landscape of BCR-ABL1 Kinase-Independent Mechanisms of Resistance to ABL1 Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia

Despite the well-established success of ABL1 tyrosine kinase inhibitors (TKIs) in the treatment of patients with chronic myeloid leukemia (CML), approximately 20% of patients treated with frontline imatinib develop resistance by 5 years on therapy. The majority (~60%) of such resistant cases are explained by acquired mutations within the BCR-ABL1 kinase domain that compromise inhibitor binding, and nearly all of these mutations are effectively targeted by one or more of the 2nd and 3rd generation ABL1 kinase inhibitors. In contrast, the remaining ~40% of imatinib-resistant cases harbor no explanatory BCR-ABL1 kinase domain mutation, presumably attributable to BCR-ABL1 kinase-independent mechanisms. We hypothesized that resistance in these patients results from acquired auxiliary molecular aberrations which persistently activate signaling pathways downstream despite inhibition of BCR-ABL1 kinase activity. To identify such mechanisms, we performed whole exome sequencing and RNA sequencing on a cohort of 135 CML patients comprising the following subgroups: newly diagnosed/TKI naïve (n=28), BCR-ABL1 kinase-dependent resistance (n=31), and BCR-ABL1 kinase-independent resistance (n=65), and TKI-induced remission (n=7). Resistant patients were required to have demonstrated clinical resistance to one or more ABL1 kinase inhibitors in the form of suboptimal response or loss of cytogenetic response; the subtype of resistance was defined based on the presence or not of an explanatory BCR-ABL1 kinase domain mutation at the time of resistance. The majority of samples collected were from patients with chronic phase CML (n=97), although smaller cohorts of accelerated phase CML, blast crisis CML, and Ph+ ALL were also profiled (n=20, 19, and 9, respectively). Among the 44,413 protein-altering and 902 splice site variants detected across the 120 WES samples, there were on average 908 missense, 146 truncation and 69 splice variants per sample. Genes with truncation and missense variants were compared between BCR-ABL1 kinase-independent and -dependent resistant chronic phase samples. A total of 44 genes were seen with a frequency difference of at least 10%, including PLEKHG5 and NKD2 (30% and 28% difference, respectively), which are involved in regulation of NF-kB and Wnt signaling. Consistent with previous reports, we also detected EZH2 and TET2 as exclusively mutated in the BCR-ABL1 kinase-independent resistance patients (6% and 3%, respectively). Further analyses stratifying variants among resistant patients according to specific ABL1 kinase inhibitor therapy failed and comparing, where available, serial samples from pre- and post-treatment for clonal expansion are underway. Additionally, sufficient material was available to perform ex vivo small-molecule inhibitor screening for 48 patient specimens, the resultant data of which was used to generate putative effective drug target profiles and integrated with exome sequencing variants to prioritize variants of functional relevance (HitWalker; Bottomly et al., Bioinformatics 2013). Among 23 patient samples exhibiting BCR-ABL1 kinase-independent resistance, the mutated genes most frequently ranked in the top 10 functional-prioritized variants were: ABL1 (which included non-kinase domain variants; 34.7%), MAP3K1, MUC4, FGF20 (each 17.4%), ARHGEF15, MEF2A, EPHA8, TYRO3, BMP2K, and IRS1 (each 13.0%). Notably, the top six candidates are members of the neutrophin (ABL1, MAP3K1, and IRS1), EPHA forward (EPHA8, ARHGEF15), and p38 MAPK signaling pathways (MAP3K1 and MEF2A). Taken together, these findings suggest that several of the same pathogenic molecular abnormalities seen in other myeloid malignancies are also present in CML patients with BCR-ABL1 kinase-independent resistance, including a subset which align to persistent re-activation of signaling pathways involved in CML disease pathogenesis and progression. As such, genetic and/or functional profiling of these patients in the clinic may translate to actionable candidates for combination therapy to maximize disease control and improve patient outcomes. DisclosuresAgarwal:CTI BioPharma Corp: Research Funding. Radich:Novartis: Consultancy, Research Funding; BMS: Consultancy; Ariad: Consultancy. Deininger:Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Druker:Pfizer: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Curis: Patents & Royalties; Array: Patents & Royalties; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; AstraZeneca: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership.

Venetoclax Resistant Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Cells Are Also Resistant to ABL Inhibitors, Which Can be Overcome By Targeting the PI3K/AKT/ mTOR Pathway

Introduction: Venetoclax is a BH3-mimetic shown to specifically inhibit BCL-2 and has activity in hematologic malignancies addicted to BCL-2, including Ph+ ALL. However, when used as a single agent resistance develops through upregulation as well as post-translational modifications of MCL-1 from a variety of different pathways. It remains unknown what effects if any BCL-2 resistance will have on ABL kinase inhibition. In these studies, we investigated whether acquired resistance to venetoclax affected kinase sensitivity in a pre clinical model of Ph+ ALL.Methods: A venetoclax resistant cell line (SUPVR2) was generated by progressively exposing the Ph+ ALL cell line SUPB15 to increasing levels of venetoclax, and then characterized to the parent cell line. Expression of both pro and anti apoptotic members of the BCL-2 family of proteins was assessed via immunoblot. Sensitivity of resistant cells was then compared to parental cells across a library of small molecule kinase inhibitors. Briefly, cells were incubated with a panel of 130 small molecule inhibitors at varying concentrations for 72 hours, and then assessed using the colorimetric MTS assay as an indirect measure of viability. Drug effectiveness was evaluated based on the IC50 of each drug on the panel against each cell line. SUPB15 and SUPVR2 cells were also exposed to increasing concentrations of dasatinib or venetoclax after treatment with 40 µM of siRNA targeting ABL-1, MCL-1 or a non-specific control (NS). Drug combinations were assessed by co-culturing resistant cells with varying concentrations of each drug in combination or as single agent, and then synergy calculations were perfromed using Calculsyn software.Results: The IC50 of the SUPVR2 cells to venetoclax was >100µM, compared to 10nM for SUPB15. Levels of MCL-1 were higher and BCL-2 were lower in SUPVR2 cells as compared to SUPB15, but no differences were seen in BCL-xL or the anti-apoptotic molecules BIM, BAX/BAK, PUMA or NOXA. When assessed with the full drug panel, SUPVR2 were found to have reduced sensitivity compared to SUPB15 to the ABL TKIs nilotinib (IC50 257.7nM vs 10.7nM ) and dasatinib (306.4nM vs 4.6nM) but not ponatinib (1.5nM vs 1nM). In contrast, SUPVR2 cells showed enhanced sensitivity to molecules that targeted the PI3K/mTOR pathway (rapamycin (0.94nM vs15.6nM), PP242 (39.4nM vs 72.5nM), INK-128 (40.6nM vs 91.0nM), BEZ235 (29nM vs 531.1nM) and PI-103 (71.3nM vs 123.3nM). Although SUPVR2 did still harbor the BCR-ABL transcript, siRNA inhibition of ABL had no impact on viability compared to SUPB15, suggesting that the SUPVR2 are no longer ABL-dependent. siRNA inhibition of MCL-1 expression restored sensitivity of the resistant cells to venetoclax, but only partially restored sensitivity to dasatinib. Co-incubation of cells with dasatinib and either rapamycin, PP-242 or PI-103 led to restoration of dasatinib sensitivity in a synergistic manner.Conclusions: Resistance to venetoclax in SUPB15 leads to concurrent resistance to ABL kinase inhibitors, which appears to occur via activation of an ABL-independent pathway. Although it is not clear whether this resistance will develop if both an ABL kinase inhibitor and a BCL-2 inhibitor are administered concurrently, our results indicate that inhibitors of PI3K/mTOR may prevent the emergence of venetoclax and ABL resistance. DisclosuresTyner:AstraZeneca: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Array Biopharma: Research Funding; Janssen Research & Development: Research Funding; Leap Oncology: Consultancy; Takeda Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Inctye: Research Funding. Druker:Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Array: Patents & Royalties; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; Pfizer: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Curis: Patents & Royalties; BMS: Research Funding; AstraZeneca: Consultancy; Array: Patents & Royalties; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties.

ABL Tyrosine Kinase Plays an Important Role in Mechanisms Involved in Genomic Instability in Multiple Myeloma

Homologous recombination (HR) is a DNA repair mechanism that uses extensive sequence homology in the participating DNA molecules for an accurate repair. In a normal cellular environment, HR is the most precise DNA repair mechanism and therefore has a unique role in the maintenance of genomic integrity and stability. Normally HR is tightly regulated, however, as it involves incision and recombination of genomic DNA fragments, if dysregulated or dysfunctional, it can also be deleterious. Consistent with this view, we have shown that elevated HR activity mediates genomic instability and development of drug resistance in MM. Here we have now investigated the mechanism that may contribute to dysregulation of HR and genomic instability in MM, as well as evaluated an agent able to decrease acquisition of new genomic changes. It has been shown that Abl kinase regulates recombinase RAD51 by affecting its expression, stability as well as phosphorylation at Y315. Phosphorylation of RAD51 (at Y315) mediates its dissociation from BCR-ABL1 kinase and migration to the nucleus to form nuclear foci, one of the initial steps in HR. We have evaluated nilotinib, a small molecule inhibitor of Abl kinase and observed that it inhibited HR activity in all MM cell lines tested, in a dose-dependent manner. At 5 µM, nilotinib inhibited HR activity in MM1S, RPMI 8226 and U266 MM cells by 64%, 78% and 80%, respectively. Nilotinib led to reduced phosphorylation of RAD51 at Y315, the phosphorylation which affects RAD51 migration. Nilotinib-mediated inhibition of RAD51 and HR activity was also associated with reduced DNA breaks, as indicated by reduced levels of g-H2AX. To determine the impact of nilotinib on genome stability, MM (RPMI 8226) cells were cultured in the presence of nilotinib for three weeks and the impact of this treatment on appearance of new copy number changes was evaluated using SNP arrays. Using day 0 cells as baseline to identify new copy number events at 3 weeks, the acquisition of new genomic changes was inhibited by 50% in the presence of nilotinib. As we have previously reported that induction of HR helps develop dexamethasone resistance in a short period of time, we investigated whether inhibition of HR by nilotinib may improve efficacy of melphalan and dexamethasone in MM. Nilotinib (at 2.5 µM) significantly increased the efficacy of melphalan (10 µM); and dexamethasone (10 nM) in RPMI 8226 cells. The relation between these observed effects and inhibition of HR is being investigated.In conclusion, we have observed that Abl tyrosine kinase plays an important role in genomic instability in myeloma and its inhibition using nilotinib, suppresses the underlying mechanism of genomic instability and reduces acquisition of new genomic changes with potential for clinical application. DisclosuresNo relevant conflicts of interest to declare.

Notch Pathway Activation Targets Leukemic Stem Cells in Chronic-Phase Chronic Myeloid Leukemia (CP-CML)

The phenomenon of disease persistence in CP-CML under therapy with ABL kinase inhibitors suggests BCR-ABL-independent mechanisms are being exploited to sustain the survival of leukemic stem cells (LSC). Many self-renewal pathways have been implicated in their survival. The role of Notch signalling has yet to be fully elucidated, with conflicting reports suggesting a variety of roles in myeloid disease. The aims of this project were (1) to assess the expression and functional role of the Notch pathway in primary CD34+CP-CML cells, and (2) to assess differences in the functional role between stem and progenitor subpopulations in disease maintenance and progression. We hypothesized that Notch activation would have different functional effects dependent on disease phase and cell maturity.Extensive analysis of existing microarray datasets, GSE47927 and E-MTAB2481, comparing CP-CML LSCs to normal HSCs, showed that the Notch pathway was downregulated in CP-CML (p=0.05, both). To validate these results, using Fluidigm technology, mRNA expression of untreated CD34+ CP-CML cells (n=24) was compared to normal bone marrow CD34+ cells (n=5). The Notch pathway was silenced in untreated CP-CML, as evidenced by downregulation of Notch target genes, HES1 (p<0.0001) and HIF1A (p=0.0029). The Notch receptor, NOTCH2, was significantly upregulated (p<0.0001) in the same samples. This was confirmed with immunofluorescence (IF) of the full-length receptor (n=5). The activated form of the Notch receptor, val1744, was absent on CD34+CP-CML cells by either IF or Western blotting (n=3), nor was there any significant change in functional analyses of CP-CML cells treated with the pharmacological Notch inhibitor, DAPT (n=5). These results support our hypothesis that the pathway is silenced in CP-CML.As CD34+ CP-CML cells abundantly express NOTCH2, we investigated whether the pathway could be reactivated through exogenous stimulation upon ligand binding. An OP9 co-culture system was utilized to allow for overexpression of Notch ligands, DLL1 or JAG1, using OP9GFP as control stroma. Compared to non-stromal conditions, culturing over 7 days on OP9GFP led to a significant increase in growth (p=0.02, n=5). The overexpression of JAG1 led to a further increase compared to OP9GFP (p=0.002). We confirmed that activation of the pathway was through JAG1, and not DLL1, by utilizing DAPT, where we observed a significant decrease in live cell counts within the OP9GFP (p=0.01) and OP9JAG1 (p=0.018) experimental arms. Upregulation of the pathway was confirmed with increased expression of HES1 at the gene level (n=4, p=0.001), andval1744 protein by IF (n=5).CP-CML samples (n=3) were FACS-sorted into Lin-CD34+38+ and Lin-CD34+38- populations to assess for functional variation in stem and progenitor populations. Following 7 days in culture, live cell counts showed a significant increase in Lin-CD34+38+ cells on OP9JAG1 co-culture (p=0.04). There was a decrease in cell growth of Lin-CD34+38- cells in the same experimental conditions (p=0.02), with an associated increased trend in late apoptotic cells (ns). We hypothesized that Notch activation would have a toxic influence on an immature CML LSC population. LTCIC assays showed a significant decrease in colonies for CD34+38-cells co-cultured with OP9JAG1 for 7 days (p<0.05, n=8).In myeloid BP (n=4), in similar experiments, sorted Lin-CD34+38+ and Lin-CD34+38- populations did not demonstrate this functional profile of Notch activation, despite an upregulation in NOTCH2 receptor expression compared to normal (n=11) (p=0.0035). To investigate this further, we sorted CP (n=12), myeloid BP (n=11), and lymphoid BP (n=5) into stem and progenitor populations, before undertaking quantitative PCR of 90 self-renewal gene components of Wnt, Notch, Hedgehog, and BMP pathways. The self-renewal pathways were highly deregulated between CP and BP. The differential gene expression in lymphoid BP was comparable to myeloid BP. There was statistically significant upregulation in Wnt components in myeloid BP compared to CP, particularly TCF7(p=0.0011). We hypothesize that Wnt upregulation is preventing Notch activation in myeloid BP-CML.Taken together, our results identify that Notch activation may be a therapeutic approach to target CP-CML LSCs. However, this cannot be translated to myeloid BP, highlighting the complexity of self-renewal pathway interaction. DisclosuresWheadon:GlaxoSmithKline: Research Funding. Holyoake:Novartis: Honoraria, Research Funding; Bristol Myers Squib: Honoraria, Research Funding. Copland:ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shire: Honoraria; Amgen: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Precision Medicine Assays Detect Novel Targetable FLT3 Fusions Amenable to Therapeutic Intervention in a Patient with Refractory Acute Myeloid Leukemia

Context:The 5-year survival rate for acute myeloid leukemia (AML) is 26.6%. The prognosis of patients with adverse events such as older age or unfavorable risk cytogenetics remains poor, even for those who undergo allogeneic hematopoietic cell transplant. AML is a heterogeneous disease and novel N-of-1 clinical trial designs may offer benefit to individuals compared to conventional clinical trials by offering improved utilization of investigational chemotherapy regimens. Precision medicine based assays that reveal a deeper understanding of the cancer biology and potential for novel therapeutics may improve survival in the future. Objective:The goal of the clinical trial on which this patient was enrolled, Individualized Treatment for Relapsed/Refractory Acute Leukemia Based on Chemosensitivity and Genomics/Gene Expression Data (ClinicalTrials.gov Identifier:NCT01872819) was to determine feasibility of a study that utilized results of comprehensive mutation analysis and an in vitrohigh throughput functional assay to choose treatment for individual patients with refractory AML. Feasibility was defined as initiating chosen treatment within 21 days. A secondary objective was to achieve a response (cytoreduction or at least partial response) greater than that expected for comparable refractory patient populations with other salvage regimens. Design, Setting, and Patient:A single center enrolled individuals who had failed at least 2 inductions at initial diagnosis or >1 salvage regimen for relapsed AML. Patients could receive any FDA approved drug or combination regimen based on molecular analysis and high throughput drug sensitivity assay. A 58 year old female was enrolled into this protocol with MECOM (EVI1) rearranged, Monosomy 7 refractory AML. Methods and Main Outcome Measures:The patient had various assays performed on her samples, including next generation sequencing and a high throughput in vitro assay that analyzed enriched blast samples for sensitivity to 150 drugs or drug combinations. MyAML™, a next generation sequencing panel, analyzed 194 genes including breakpoint hotspot loci with long paired end sequencing and high depth that optimized detection of large insertion and deletions and other structural variants found in AML at low variant allele frequency.Results:The MyAML assay detected multiple variants including: NRAS:c.38G>A; p.Gly13Asp VAF = 100%, RUNX1:c.494_495ins; p.165_R166ins VAF = 42%, WT1:c.1149_1150ins; p.E384Pfs*5 VAF=38%. Fusions were also detected: t(13;17)(q12.2;q11.2), t(8;13)(q21.13;q12.2), and t(9;12)(q32;p13.2) which involved FLT3 novel fusions. FLT3 internal tandem duplications (ITD) or tyrosine kinase domain (TKD) variants were not detected. The assay also detected the Monosomy 7 and confirmed the t(2;3) as a THADA-MECOM (EVI1) fusion to the nucleotide breakpoint. These variants involved activated signaling, myeloid transcription factor and DNA demethylation pathways.The high throughput drug sensitivity assay identified sensitivity to kinase inhibitors such as the MEK inhibitor selumetinib (IC50 8.1 X 10e-9 M), Flt3 inhibitor staurosporine (IC50 1.4 X 10e-8 M) and Abl kinase inhibitor ponatinib (IC50 2.7 X 10e-8 M). Insurance coverage for these agents was not able to be obtained as this indication would be considered off label. However, based on the FLT3 fusion we identified using MyAML, we decided to treat the patient with the tyrosine kinase inhibitor sorafenib that we were able to obtain through a patient assistance program.This novel case is the first demonstration of a FLT3 fusion detection with a drug sensitivity assay suggesting that kinase inhibitors with multiple targets might be suitable for this type of variant.Conclusion:Specialized assays designed to identify clonal and subclonal architecture of genes associated with specific diseases can reveal variants that present therapeutic options not currently utilized for high risk patients, suggesting broader use of this approach could improve current clinical outcomes. DisclosuresPatay:Invivoscribe, Inc: Consultancy. Carson:Invivoscribe, Inc: Employment. Becker:GlycoMimetics: Research Funding.

α3β1 Integrin Suppresses Prostate Cancer Metastasis via Regulation of the Hippo Pathway.

Existing anticancer strategies focused on disrupting integrin functions in tumor cells or tumor-involved endothelial cells have met limited success. An alternative strategy is to augment integrin-mediated pathways that suppress tumor progression, but how integrins can signal to restrain malignant behavior remains unclear. To address this issue, we generated an in vivo model of prostate cancer metastasis via depletion of α3β1 integrin, a correlation observed in a significant proportion of prostate cancers. Our data describe a mechanism whereby α3β1 signals through Abl family kinases to restrain Rho GTPase activity, support Hippo pathway suppressor functions, and restrain prostate cancer migration, invasion, and anchorage-independent growth. This α3β1-Abl kinase-Hippo suppressor pathway identified α3 integrin-deficient prostate cancers as potential candidates for Hippo-targeted therapies currently under development, suggesting new strategies for targeting metastatic prostate cancer based on integrin expression. Our data also revealed paradoxical tumor suppressor functions for Abl kinases in prostate cancer that may help to explain the failure of Abl kinase inhibitor imatinib in prostate cancer clinical trials. Cancer Res; 76(22); 6577-87. ©2016 AACR.

Initial testing of VS-4718, a novel inhibitor of focal adhesion kinase (FAK), against pediatric tumor models by the Pediatric Preclinical Testing Program.

VS-4718, a novel inhibitor of focal adhesion kinase (FAK), was tested against the Pediatric Preclinical Testing Program's (PPTP's) in vitro cell line panel and showed a median relative IC50 of 1.22 μM. VS-4718 was tested in vivo against the PPTP xenograft models using a dose of 50 mg/kg administered by the oral route twice daily for 21 days. VS-4718 induced significant differences in an event-free survival distribution compared with control in 18 of 36 of the evaluable solid tumor xenografts and in 0 of 8 acute lymphoblastic leukemia (ALL) xenografts, but no xenograft lines showed tumor regression. Future plans include further evaluation of the role of FAK inhibition in combination with ABL kinase inhibitors for Ph+ ALL.

Opening the door to the development of novel Abl kinase inhibitors.

The discovery of the importance of kinase activity and its relationship to the emergence and proliferation of cancer cells, due to changes in normal physiology, opened a remarkable pathway for the treatment of chronic myelogenous leukemia through intense search of drug candidates. Six Abl kinase inhibitors have received the US FDA approval as chronic myelogenous leukemia treatment, and continuous efforts in obtaining new, more effective and selective molecules are being carried out. Herein we discuss the mechanisms of Abl inhibition, structural features and ligand/protein interactions that are important for the design of new Abl kinase inhibitors. This review provides a broad overview of binding mode predictions, through molecular docking, which can be an approach to discover novel Abl kinase inhibitors.

Design, synthesis, and biological activity of 4-(imidazo[1,2-b]pyridazin-3-yl)-1H-pyrazol-1-yl-phenylbenzamide derivatives as BCR–ABL kinase inhibitors

A series of 4-((pyrazolo[1,5-a]pyrimidin-6-yl)-1H-pyrazol-1-yl)phenyl-3-benzamide derivatives and 4-((imidazo[1,2-b]pyridazin-3-yl)-1H-pyrazol-1-yl-)phenyl-3-benzamide derivatives were designed, synthesized as new BCR–ABL tyrosine kinase inhibitors by using combinational strategies of scaffold hopping and conformational constraint. These new compounds were screened for BCR–ABL1 kinase inhibitory activity, and most of them appeared good inhibitory activity against BCR–ABL1 kinase. One of the most potent compounds 16a strongly suppressed BCR–ABL1 kinase with IC50 value of 8.5nM. The tested compounds 16a and 16i showed strong inhibitory activities against K562 with IC50 value of less than 2nM. Molecular docking studies indicated that these compounds fitted well with the active site of BCR–ABL1 protein. The results showed these inhibitors may serve as lead compounds for further developing new drugs targeted BCR–ABL kinase.

CagA Phosphorylation in Helicobacter pylori-Infected B Cells Is Mediated by the Nonreceptor Tyrosine Kinases of the Src and Abl Families.

CagA is one of the most important virulence factors of the human pathogen Helicobacter pylori. CagA expression can be associated with the induction of severe gastric disorders such as gastritis, ulceration, gastric cancer, or mucosa-associated lymphoid tissue (MALT) lymphoma. After translocation through a type IV secretion system into epithelial cells, CagA is tyrosine phosphorylated by kinases of the Src and Abl families, leading to drastic cell elongation and motility. While the functional role of CagA in epithelial cells is well investigated, knowledge about CagA phosphorylation and its associated signal transduction pathways in B cells is only marginal. Here, we established the B cell line MEC1 derived from a B cell chronic lymphocytic leukemia (B-CLL) patient as a new infection model to study the signal transduction in B cells controlled by H. pylori. We observed that CagA was rapidly injected, strongly tyrosine phosphorylated, and cleaved into a 100-kDa N-terminal and a 40-kDa C-terminal fragment. To identify upstream signal transduction pathways of CagA phosphorylation in MEC1 cells, pharmacological inhibitors were employed to specifically target Src and Abl kinases. We observed that CagA phosphorylation was strongly inhibited upon treatment with an Src inhibitor and slightly diminished when the Abl kinase inhibitor imatinib mesylate (Gleevec) was applied. The addition of dasatinib to block c-Abl and Src kinases led to a complete loss of CagA phosphorylation. In conclusion, these results demonstrate an important role for Src and Abl tyrosine kinases in CagA phosphorylation in B cells, which represent druggable targets in H. pylori-mediated gastric MALT lymphoma.