ABL Kinase Inhibitor Imatinib Research Articles

ABL Genomic Editing Sufficiently Abolishes Oncogenesis of Human Chronic Myeloid Leukemia Cells In Vitro and In Vivo.

Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target the BCR-ABL junction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety of BCR-ABL junctions in CML patients, we utilized gene editing of the human ABL gene for clinical applications. Using the ABL gene-edited virus in K562 cells, we detected 41.2% indels in ABL sgRNA_2-infected cells. The ABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced the ABL gene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population in ABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with the ABL gene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using the ABL gene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of the ABL gene in maintaining CML cell survival and tumorigenicity in vitro and in vivo. ABL gene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.

The ABL kinase inhibitor imatinib causes phenotypic changes and lethality in adult Schistosoma japonicum.

Schistosomiasis caused by different species of schistosome parasites is one of the most debilitating helminthic diseases of humans worldwide. For decades, chemotherapy is the main method of controlling schistosomiasis. However, the fear of drug resistance has motivated the search for alternatives. It has been demonstrated that the ABL kinase inhibitor imatinib affected the development and survival of Schistosoma mansoni in vitro; however, there is still lack of information on whether imatinib also affects other schistosome species such as Schistosoma japonicum. In the present study, the anti-schistosomal potency of imatinib on adult S. japonicum was investigated in vitro, and the results showed that imatinib had a significant impact on various physiological processes of S. japonicum adult worms. Besides its negative effects on worm motility, pairing stability, and gonad development, imatinib caused pathological changes in the gastrodermis as well as the death of the parasite. Our findings suggest that imatinib is an intriguing candidate for further development as an option to fight S. japonicum.

Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.

The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.

The vascular endothelial specific IL-4 receptor alpha–ABL1 kinase signaling axis regulates the severity of IgE-mediated anaphylactic reactions

Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)-mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. Mice deficient in VE IL-4Rα and models of passive and active oral antigen- and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4- and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1-dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions.

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.

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.

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.

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.

Abl Family Kinases Differentially Regulate LPS‐induced Endothelial Barrier Dysfunction

Rationale: Acute Lung Injury (ALI) involves inflammation-induced disruption of the pulmonary endothelial cell (EC) barrier. Abl family kinases, c-Abl and Arg, have distinct roles in barrier regulation in response to thrombin, histamine, VEGF and oxidative stress. Our prior work indicates that the Abl kinase inhibitor imatinib protects against LPS-induced ALI. To elucidate the underlying mechanisms, we investigated the roles of c-Abl and Arg in LPS-induced EC dysfunction. Methods Intercellular gap formation was quantified in human pulmonary artery ECs treated with imatinib (20 μM) and LPS (1 μg/mL) via FITC-avidin binding to biotinylated gelatin. Western blots and immunofluorescence microscopy were conducted on LPS challenged ECs to assess c-Abl/Arg expression, kinase activity (pCrkL), and sub-cellular localization. Additionally, siRNA was used to assess the roles of c-Abl/Arg in EC contractile apparatus localization and LPS-induced EC activation (VCAM-1, IL-8). Results C-Abl/Arg inhibition attenuated LPS-induced gap formation (22%, p=0.01). LPS increased c-Abl/Arg activation, without altering expression. However, only c-Abl co-localized with actin stress fibers after LPS. Additionally, only c-Abl silencing attenuated LPS-induced VCAM-1 expression (33%, p=0.04) and IL-8 release (48%, p=0.048). Furthermore, the actinomyosin contractile apparatus was localized centrally in c-Abl-silenced ECs, but peripherally in Arg-silenced ECs. Conclusions Abl family kinases differentially regulate EC dysfunction in response to LPS. The FDA approved inhibitors of these kinases may represent potential therapeutic options for ALI.

Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells.

BackgroundThe ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2.MethodsWe investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5).ResultsImatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348.ConclusionsThese results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells.

Evidence for a role of p130Cas in JAM‐A‐dependent signaling events (60.3)

Junctional adhesion molecule‐A (JAM‐A) is a tight junction protein that plays a key role in regulation of intestinal mucosal homeostasis. Previous studies have shown that loss of JAM‐A decreases beta‐1 integrin‐mediated cell migration and increases Akt‐dependent proliferation however signaling pathways downstream of JAM‐A remain incompletely defined. Levels of known actin‐dependent regulators of cell migration and proliferation in epithelial cells derived from JAM‐A deficient mice and ex vivo cultures of small intestinal enteroids were thus examined. We observed dramatic changes in p130Cas, a critical signaling component of focal adhesions that is regulated by ABL kinase. Western blots of JAM‐A deficient intestinal epithelial cells (IEC) and enteroids revealed significantly decreased levels of phosphorylated p130Cas. In addition, JAM‐A deficient colonic IEC showed decreased levels of the migration‐regulating integrin beta‐1 in parallel with decreased E‐cadherin, a negative regulator of proliferation. Treatment of cultured IEC with the ABL kinase inhibitor Imatinib resulted in delayed epithelial wound closure, inhibited cell spreading and decreased p130Cas phosphorylation at Y410 similar to that observed with JAM‐A deficiency. These findings suggest that p130Cas may act to transduce JAM‐A‐dependent signals to beta‐1 integrin to regulate migration, and E‐cadherin to inhibit proliferation.Grant Funding Source: Supported by NIH T32 DK007771‐12, Pathobiology of Mucosal/Epithelial Disease

DNA damage-activated ABL-MyoD signaling contributes to DNA repair in skeletal myoblasts

Previous works have established a unique function of MyoD in the control of muscle gene expression during DNA damage response in myoblasts. Phosphorylation by DNA damage-activated ABL tyrosine kinase transiently inhibits MyoD-dependent activation of transcription in response to genotoxic stress. We show here that ABL-MyoD signaling is also an essential component of the DNA repair machinery in myoblasts exposed to genotoxic stress. DNA damage promoted the recruitment of MyoD to phosphorylated Nbs1 (pNbs1)-containing repair foci, and this effect was abrogated by either ABL knockdown or the ABL kinase inhibitor imatinib. Upon DNA damage, MyoD and pNbs1 were detected on the chromatin to MyoD target genes without activating transcription. DNA damage-mediated tyrosine phosphorylation was required for MyoD recruitment to target genes, as the ABL phosphorylation-resistant MyoD mutant (MyoD Y30F) failed to bind the chromatin following DNA damage, while retaining the ability to activate transcription in response to differentiation signals. Moreover, MyoD Y30F exhibited an impaired ability to promote repair in a heterologous system, as compared with MyoD wild type (WT). Consistently, MyoD-null satellite cells (SCs) displayed impaired DNA repair that was rescued by reintroduction of MyoD WT but not by MyoD Y30F. In addition, inhibition of ABL kinase prevented MyoD WT-mediated rescue of DNA repair in MyoD-null SCs. These results identify an unprecedented contribution of MyoD to DNA repair and suggest that ABL-MyoD signaling coordinates DNA repair and transcription in myoblasts.

EIF4B Phosphorylation by Pim Kinases Plays a Critical Role in Cellular Transformation byAblOncogenes

Alterations in translation occur in cancer cells, but the precise pathogenic processes and mechanistic underpinnings are not well understood. In this study, we report that interactions between Pim family kinases and the translation initiation factor eIF4B are critical for Abl oncogenicity. Pim kinases, Pim-1 and Pim-2, both directly phosphorylated eIF4B on Ser406 and Ser422. Phosphorylation of eIF4B on Ser422 was highly sensitive to pharmacologic or RNA interference-mediated inhibition of Pim kinases. Expression and phosphorylation of eIF4B relied upon Abl kinase activity in both v-Abl- and Bcr-Abl-expressing leukemic cells based on their blockade by the Abl kinase inhibitor imatinib. Ectopic expression of phosphomimetic mutants of eIF4B conferred resistance to apoptosis by the Pim kinase inhibitor SMI-4a in Abl-transformed cells. In contrast, silencing eIF4B sensitized Abl-transformed cells to imatinib-induced apoptosis and also inhibited their growth as engrafted tumors in nude mice. Extending these observations, we found that primary bone marrow cells derived from eIF4B-knockdown transgenic mice were less susceptible to Abl transformation, relative to cells from wild-type mice. Taken together, our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes, and they highlight eIF4B as a candidate therapeutic target for treatment of Abl-induced cancers.

Photocleavable peptide–oligonucleotide conjugates for protein kinase assays by MALDI-TOF MS

Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with the potential to help overcome these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptide-oligonucleotide conjugates. After washing away contaminants and following photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate-oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC(50) for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics.

Abstract 1064: Pim serine/threonine kinases regulate the phosphorylation and function of eIF4B protein

Abstract Eukaryotic initiation factor 4B (eIF4B) is a multidomain protein with a critical role in the initiation of protein synthesis, and has long been known as a hyperphosphorylated protein. It has been thought that function of eIF4B can be regulated by phosphorylation. The Pim family of Ser/Thr kinase, which is consisted of three members: Pim-1, Pim-2, and Pim-3, has been implicated in multiple cellular processes including cellular survival, anti-apoptosis and tumorigenesis. In this study, we identified eIF4B as a Pim kinase substrate. We found that Pim-1, Pim-2 but not Pim-3 can upregulate phosphorylation levels of eIF4B on Ser406 and Ser422 using 293T cell system, while kinase-dead mutants of Pim-1 or Pim-2 significantly inhibit phosphorylation of eIF4B. Furthermore, in vitro kinase assay revealed that Pim-1 kinase can directly phosphorylate eIF4B on Ser422. As expected, we found that Pim-1 kinase directly bound to eIF4B using GST-pull down assay and co-immunoprecipitation experiments. Interestingly, levels of eIF4B protein in v-Abl transformants treated with the Abl kinase inhibitor imatinib decreased markedly, indicating that expression of eIF4B is Abl kinase dependant. Our results suggest that Pim kinases regulate cellular processes via phosphorylating downstream eIF4B, and thereby alter the function of eIF4B in v-Abl transformants. Together, these data possibly provide new insights into molecular mechanisms of Abl oncogene-mediated cellular transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1064. doi:10.1158/1538-7445.AM2011-1064

Rapid identification of therapeutic targets in hematologic malignancies via functional genomics

The clinical application of gene-targeted drugs has transformed cancer therapy. The hallmark example of this strategy is use of the ABL kinase inhibitor imatinib for treatment of patients with chronic myeloid leukemia (CML). This remarkable clinical success has also stimulated an expansive search for personalized gene targets in all patients to facilitate broad application of targeted therapy for cancer. However, achievement of this objective will require simultaneous work towards several complementary goals. The first step towards broad application of gene-targeted therapy must entail a rapid means to identify target oncogenes in individual patients. Next, we must identify well-tolerated, gene-specific drugs that are collectively effective against a wide diversity of gene targets. Finally, we must develop protocols by which individual patients are matched with appropriate, gene-targeted drugs in a clinically relevant time frame. While these may seem like difficult tasks, we are fortunate to have a wide variety of new and rapidly evolving research tools at our disposal. These include next-generation sequencing of the genome and transcriptome, single nucleotide polymorphism (SNP)/copy number variations (CNV) and gene expression microarrays, and RNAi libraries for the application of functional screens. In this review we discuss the advantages and disadvantages of each of these techniques with the goal of demonstrating that no single technique will be sufficient as a standalone technology, but rather it will be the integration of all techniques that will enable broad application of gene-targeted cancer therapies.

Dominant Negative Effect of Palmitoylation-Deficient NRAS In Suppression of BCR/ABL Leukemogenesis

AbstractAbstract 3157RAS mutations are found in ∼30% of all human cancers, with NRAS being the RAS isoform found most frequently mutated in hematological malignancies including acute myelogenous leukemia (AML) and chronic myelomonocytic leukemia (CMML). We have previously shown that expression of oncogenic NRAS efficiently induces a CMML- or AML-like disease in mice. Like all RAS proteins, NRAS must undergo a series of post-translational modifications for differential targeting to distinct cellular membrane microdomains. We have previously found that palmitoylation, in addition to prenylation, is an essential process for NRAS leukemogenesis. The results suggest that targeting RAS palmitoylation may be an effective therapy for hematological malignancies as well as other NRAS related cancers.In addition to the direct activation by mutations, RAS can also be functionally activated by other oncogenic mutations, including activated protein tyrosine kinases such as BCR/ABL. While significant advances have been made in the treatment of chronic myelogenous leukemia (CML) by targeting the kinase activity of BCR/ABL, these treatments do not constitute a cure, as BCR/ABL-positive cells persist and eventually develop mutations that cause resistance to the kinase inhibitors. In addition, although advances have also been achieved in the treatment of BCR/ABL+ B-acute lymphoblastic lymphoma (B-ALL) through the use of combination chemotherapy along with the ABL kinase inhibitor imatinib, BCR/ABL+ B-ALL continues to pose a huge challenge; they are highly refractory to imatinib, and are still the least treatable subtype of ALL. The identification of molecular targets that impede the progression of BCR/ABL+ leukemias not relying on targeting the kinase activity of BCR/ABL is likely beneficial. Previous studies have shown that RAS is a critical downstream transducer of the oncogenic signaling by BCR/ABL. We have shown that palmitoylation-deficient oncogenic NRAS is mislocalized away from the plasma membrane yet still capable of binding GTP in cells. This finding suggests that palmitoylation-deficient, activated RAS may have a dominant negative effect in RAS signaling. As a proof-of-concept study for treating cancers driven by RAS regulators through blocking RAS palmitoylation, we evaluated the effect of blocking RAS palmitoylation in BCR/ABL leukemogenesis. We found that expression of palmitoylation-deficient oncogenic NRAS or palmitoylation-deficient wild type NRAS significantly impeded progression of BCR/ABL induced B-ALL- and CML-like diseases in mice. The palmitoylation-deficient NRAS suppresses the activation of downstream effectors of RAS, possibly by sequestrating RAS effectors away from the plasma membrane. The results suggest that targeting RAS palmitoylation may also constitute an effective therapy in cancers driven by oncogenes upstream of RAS. Disclosures:No relevant conflicts of interest to declare.

ABL fusion oncogene transformation and inhibitor sensitivity are mediated by the cellular regulator RIN1

ABL gene translocations create constitutively active tyrosine kinases that are causative in chronic myeloid leukemia, acute lymphocytic leukemia and other hematopoietic malignancies. Consistent retention of ABL SH3/SH2 autoinhibitory domains, however, suggests that these leukemogenic tyrosine kinase fusion proteins remain subject to regulation. We resolve this paradox, demonstrating that BCR-ABL1 kinase activity is regulated by RIN1, an ABL SH3/SH2 binding protein. BCR-ABL1 activity was increased by RIN1 overexpression and decreased by RIN1 silencing. Moreover, Rin1−/− bone marrow cells were not transformed by BCR-ABL1, ETV6-ABL1 or BCR-ABL1T315I, a patient-derived drug-resistant mutant, as judged by growth factor independence. Rescue by ectopic RIN1 verified a cell autonomous mechanism of collaboration with BCR-ABL1 during transformation. Sensitivity to the ABL kinase inhibitor imatinib was increased by RIN1 silencing, consistent with RIN1 stabilization of an activated BCR-ABL1 conformation having reduced drug affinity. The dependence on activation by RIN1 to unleash full catalytic and cell transformation potential reveals a previously unknown vulnerability that could be exploited for treatment of leukemic cases driven by ABL translocations. The findings suggest that RIN1 targeting could be efficacious for imatinib-resistant disease and might complement ABL kinase inhibitors in first-line therapy.

Abstract 632: Cross-resistance of myeloid cells transformed with Bcr-Abl imatinib-resistant mutant T315I to Src-family kinase inhibitors

Abstract Bcr-Abl, the constitutively active protein-tyrosine kinase arising from the Philadelphia chromosome translocation, plays a key role in the etiology of chronic myelogenous leukemia (CML). Treatment of CML with the Abl kinase inhibitor imatinib has been a clinical success in the chronic phase of the disease. However, treatment failure in the blast crisis phase is attributable to imatinib resistance caused by point mutations in the kinase domain of Bcr-Abl. One of the most clinically challenging resistance mutations involves substitution of the gatekeeper threonine with isoleucine (T315I). This residue is located within the ATP-binding site and is essential for imatinib binding. Src family kinases (SFKs) are important mediators of Bcr-Abl signal transduction and oncogenesis in CML. Bcr-Abl binds to and activates multiple SFKs in CML cells, including Hck and Lyn. In turn, active SFKs directly phosphorylate regulatory tyrosine residues in the SH3 domain, the SH2-kinase linker, and the Bcr-derived portion of Bcr-Abl. Previous work from our group showed that global inhibition of SFK activity with the ATP-competitive pyrrolo-pyrimidine A-419259 leads to growth arrest and apoptosis in CML cell lines and in primary CD34+ CML cells. In addition, expression of an Hck mutant with engineered resistance to A-419259 protects CML cells against the antiproliferative and apoptotic effects of this compound. Other studies also correlate CML progression and drug resistance with overexpression and activation of Hck and Lyn. Given the important role of SFKs in Bcr-Abl-mediated signaling and drug resistance, we studied the effect of A-419259 on human TF-1 myeloid cells transformed with clinically relevant imatinib-resistant forms of Bcr-Abl (E255V, Y253H and T315I). Proliferation of TF-1 cells transformed with Bcr-Abl E255V and Y253H was markedly inhibited by A-419249. The anti-proliferative effects of the inhibitor correlated with suppression of overall SFK activity. However, TF-1 cells transformed with the gatekeeper mutant T315I were completely cross-resistant to the SFK inhibitor A-419259. Surprisingly, SFK activity in TF-1 cells expressing Bcr-Abl T315I was not inhibited by this compound. Immunoprecipitation experiments show that endogenous Lyn remains active in A-419259-treated TF-1 cells transformed with Bcr-Abl T315I. In addition, over-expressed Hck remains active in TF-1/T315I cells upon A-419259 treatment, but is completely inhibited in TF-1 cells transformed with wild-type Bcr-Abl. These results suggest that in general, SFKs are important targets to overcome imatinib resistance in CML. However, our data also suggest a new mechanism of Bcr-Abl T315I-induced resistance to drugs that target SFKs in CML. We speculate that this effect may be due to an acquired ability of Bcr-Abl T315I to phosphorylate SFKs at novel sites that interfere with drug action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 632.

Effects of Bosutinib (SKI-606) in CML: Kinase Target Profile, Effects on BCR/ABL Mutants, and Synergism with Dasatinib in T315I+ Cells

Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50>1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50<0.5 nM) and most of the ABL mutants tested (H396P, M351T, Q252H, and Y253F) were all completely inhibited by both drugs at 1 μM in the kinase assay, the ABL T315I mutant was inhibited by bosutinib (IC50=26 nM) almost 70 times more potently than by dasatinib. Together, these data show that bosutinib and dasatinib synergize with each other in producing antileukemic effects on CML cells including BCR/ABL T315I+ subclones. These synergistic effects may be explained by differential target kinase profiles and by the fact that bosutinib retains some activity against the BCR/ABL T315I mutant kinase.