Dasatinib Sensitivity Research Articles

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

AbstractThe t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Abstract 2521: New predictive markers for dasatinib responsiveness in triple-negative breast cancer: A biomarker discovery study

Abstract Triple negative breast cancer is the most heterogeneous and aggressive type of breast tumor type and is characterized by the lack of expression of clinical biomarkers (ER, PR, and HER2) and targeted therapies. In this regard, different clinical trials have failed, unable to stratify these patients to find an effective response to specific therapy. This study aimed to identify biomarkers exclusively expressed in the basal mammary compartment that can help us to subclassify the triple-negative breast cancer subtype. Based on computational analysis from single-cell RNA sequencing data, we have identified a list of basal identity-associated genes (BC-markers). Subsequent histological validation confirmed the expression pattern of these markers in a cohort of 50 samples, which has allowed us to stratify triple-negative breast cancer patients into BC-TNBC and luminal-like subtypes. Importantly, the expression of these markers correlated with poorer prognosis in TNBC patients. Functional analysis revealed that TAGLN played a significant role in cell proliferation and migration, potentially impacting tumor aggressiveness. Moreover, the expression of these BC-markers is associated with a mesenchymal phenotype which corresponds to the previously described Lehmannn’s mesenchymal-like signature. In this study, TAGLN appeared to influence dasatinib sensitivity, suggesting its potential as a predictive biomarker for dasatinib response in TNBC patients. Citation Format: Daniel Ortega-Álvarez, David Tebar, Marta Casado, Elena Castillo, Cristina Guardia, David Olivares-Osuna, Eva Musulén, Elisabetta Mereu, Ginés Luengo, Eva M. Galán-Moya, Veronica Rodilla. New predictive markers for dasatinib responsiveness in triple-negative breast cancer: A biomarker discovery study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2521.

Genetic and Pharmacological Inhibition of BTK Increases Dasatinib Sensitivity in Vitro and In Vivo Including CNS-Infiltrating E2A-PBX1+/Pre-BCR+ ALL Cells

Genetic and Pharmacological Inhibition of BTK Increases Dasatinib Sensitivity in Vitro and In Vivo Including CNS-Infiltrating E2A-PBX1+/Pre-BCR+ ALL Cells

An integrative gene expression signature analysis identifies CMS4 KRAS-mutated colorectal cancers sensitive to combined MEK and SRC targeted therapy

BackgroundOver half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s).MethodsWe classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi “bypass” resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154).ResultsHere we report a central role of SRC in mediating “bypass”-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P < 10–320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs.ConclusionsLarge human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node–-an Achilles’ heel–-in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Dynamic expression of SNAI2 in prostate cancer predicts tumor progression and drug sensitivity.

Prostate cancer is a highly heterogeneous disease, understanding the crosstalk between complex genomic and epigenomic alterations will aid in developing targeted therapeutics. We demonstrate that, even though snail family transcriptional repressor 2 (SNAI2) is frequently amplified in prostate cancer, it is epigenetically silenced in this disease, with dynamic changes in SNAI2 levels showing distinct clinical relevance. Integrative clinical data from 18 prostate cancer cohorts and experimental evidence showed that gene fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2–ERG fusion) is involved in the silencing of SNAI2. We created a silencer score to evaluate epigenetic repression of SNAI2, which can be reversed by treatment with DNA methyltransferase inhibitors and histone deacetylase inhibitors. Silencing of SNAI2 facilitated tumor cell proliferation and luminal differentiation. Furthermore, SNAI2 has a major influence on the tumor microenvironment by reactivating tumor stroma and creating an immunosuppressive microenvironment in prostate cancer. Importantly, SNAI2 expression levels in part determine sensitivity to the cancer drugs dasatinib and panobinostat. For the first time, we defined the distinct clinical relevance of SNAI2 expression at different disease stages. We elucidated how epigenetic silencing of SNAI2 controls the dynamic changes of SNAI2 expression that are essential for tumor initiation and progression and discovered that restoring SNAI2 expression by treatment with panobinostat enhances dasatinib sensitivity, indicating a new therapeutic strategy for prostate cancer.

Network-based systems pharmacology reveals heterogeneity in LCK and BCL2 signaling and therapeutic sensitivity of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and novel therapeutics are much needed. Profiling patient leukemia' drug sensitivities ex vivo, we discovered that 44.4% of childhood and 16.7% of adult T-ALL cases exquisitely respond to dasatinib. Applying network-based systems pharmacology analyses to examine signal circuitry, we identified preTCR-LCK activation as the driver of dasatinib sensitivity, and T-ALL-specific LCK dependency was confirmed in genome-wide CRISPR-Cas9 screens. Dasatinib-sensitive T-ALLs exhibited high BCL-XL and low BCL2 activity and venetoclax resistance. Discordant sensitivity of T-ALL to dasatinib and venetoclax is strongly correlated with T-cell differentiation, particularly with the dynamic shift in LCK vs. BCL2 activation. Finally, single-cell analysis identified leukemia heterogeneity in LCK and BCL2 signaling and T-cell maturation stage, consistent with dasatinib response. In conclusion, our results indicate that developmental arrest in T-ALL drives differential activation of preTCR-LCK and BCL2 signaling in this leukemia, providing unique opportunities for targeted therapy.

Targeting EGFR exon 20 insertions in non-small cell lung cancer by exploiting a dependency on parallel SRC signalling

Abstract Background Inframe insertions in exon 20 of the epidermal growth factor receptor (EGFR) gene are oncogenic drivers in non-small cell lung cancer (NSCLC). However, unlike more common EGFR mutations, low frequency EGFR exon 20 insertions are resistant to clinically approved EGFR inhibitors and are associated with poor patient prognosis. There is an unmet clinical need to identify novel therapies that will be effective for patients with EGFR exon 20 insertions. Methods Cell viability of three patient-derived NSCLC cell line models harbouring EGFR exon 20 insertions, CUTO14 (A767_V769dupASV), CUTO17 (N771_H773dupNPH) and CUTO18 (S768_D770dupSVD) was assessed after treatment with a chemical compound screen targeting cancer-associated pathways. Western blotting was used to probe cell signalling following dasatinib treatment. Mutant SRC expression constructs were introduced into cell lines via lentiviral transduction and depletion of endogenous SRC levels was achieved using RNAi. Models of acquired drug resistance were developed by long-term drug exposure. Results Out of 58 screened compounds, the tyrosine kinase inhibitor dasatinib was a shared hit across all EGFR exon 20 insertion models and was superior to the EGFR inhibitor gefitinib in cell viability and apoptosis assays. Dasatinib sensitivity was rescued by expression of a gatekeeper mutant SRC that does not bind dasatinib, whilst all models were sensitive to RNAi-mediated depletion of endogenous SRC expression. Importantly, models of acquired dasatinib resistance maintain sensitivity to poziotinib, an EGFR inhibitor that can target EGFR exon 20 insertions. Similarly, dasatinib effectively inhibits cell viability of a model of acquired resistance to poziotinib, indicating two independent, targetable signalling nodes. Combined treatment with dasatinib and poziotinib prevented the emergence of drug resistance. Conclusions EGFR exon 20 insertion lung cancer models are sensitive to inhibition of parallel SRC signalling via dasatinib treatment. In a patient population with limited therapeutic options, an upfront rational combination of dasatinib with EGFR inhibitors that target EGFR exon 20 insertions has the potential to achieve durable responses. Legal entity responsible for the study Paul Huang. Funding Institute of Cancer Research, Cancer Research UK. Disclosure R. Doebele: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses, Licensing / Royalties: Genentech/Roche; Advisory / Consultancy, Travel / Accommodation / Expenses, Licensing / Royalties: Ignyta; Licensing / Royalties: Foundation Medicine; Advisory / Consultancy, Licensing / Royalties: Loxo Oncology; Advisory / Consultancy: Bayer; Advisory / Consultancy, Shareholder / Stockholder / Stock options, Licensing / Royalties: Rain Therapeutics; Honoraria (self), Advisory / Consultancy: Takeda/Millenium; Licensing / Royalties: Abbot Molecular. All other authors have declared no conflicts of interest.

CBP Modulates Sensitivity to Dasatinib in Pre-BCR+ Acute Lymphoblastic Leukemia.

Dasatinib is a multi-tyrosine kinase inhibitor approved for treatment of Ph+ acute lymphoblastic leukemia (ALL), but its efficacy is limited by resistance. Recent preclinical studies suggest that dasatinib may be a candidate therapy in additional ALL subtypes including pre-BCR+ ALL. Here we utilized shRNA library screening and global transcriptomic analysis to identify several novel genes and pathways that may enhance dasatinib efficacy or mitigate potential resistance in human pre-BCR+ ALL. Depletion of the transcriptional coactivator CBP increased dasatinib sensitivity by downregulating transcription of the pre-BCR signaling pathway previously associated with dasatinib sensitivity. Acquired resistance was due, in part, to upregulation of alternative pathways including WNT through a mechanism, suggesting transcriptional plasticity. Small molecules that disrupt CBP interactions with the CREB KID domain or β-catenin showed promising preclinical efficacy in combination with dasatinib. These findings highlight novel modulators of sensitivity to targeted therapies in human pre-BCR+ ALL, which can be reversed by small-molecule inhibitors. They also identify promising therapeutic approaches to ameliorate dasatinib sensitivity and prevent resistance in ALL.Significance: These findings reveal mechanisms that modulate sensitivity to dasatinib and suggest therapeutic strategies to improve the outcome of patients with acute lymphoblastic leukemia.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/22/6497/F1.large.jpg Cancer Res; 78(22); 6497-508. ©2018 AACR.

Dasatinib promotes TRAIL-mediated apoptosis by upregulating CHOP-dependent death receptor 5 in gastric cancer.

Dasatinib, a tyrosine kinase inhibitor, has been approved for first‐line treatment of leukemia and has also been evaluated for use in numerous other cancers. However, its role in gastric cancer (GC) remains unclear. Therefore, the aim of this study was to investigate how dasatinib suppresses the growth of GC cells and interacts with chemotherapeutic drugs. The results showed that, in the presence of dasatinib, proliferation of GC cells decreased and apoptosis increased, and that Fas‐associated death domain protein and caspase‐8 are essential to dasatinib‐induced cell apoptosis in GC. In addition, we found that dasatinib increased the expression of death receptor 5 (DR5) in GC cells. Dasatinib enhanced apoptosis induced by tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) in GC cells. Moreover, increased DR5 expression facilitated dasatinib‐induced apoptosis; the dasatinib‐induced increase in DR5 expression was mediated by CCAAT/enhancer‐binding protein homologous protein (CHOP). Furthermore, dasatinib also synergized with TRAIL to induce apoptosis via DR5 in GC cells. Our results show that dasatinib promoted TRAIL‐mediated apoptosis via upregulation of CHOP‐dependent DR5 expression in GC, suggesting that DR5 induction can be used as an indicator of dasatinib sensitivity.

PTPN11 Mutations in JMML Confer Dasatinib Sensitivity in a TNK2-Dependent Manner

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare disease characterized by an overproduction of granulocytes and monocytes, with cells displaying hypersensitivity to stimulation by the cytokine GM-CSF in culture. The only curative approach for JMML is allogeneic hematopoietic stem cell transplant, and disease recurrence is the leading cause of death in these patients, occurring within two years in 50% of patients. New therapeutic strategies are needed to improve treatment and survival.Methods: In order to better identify therapeutic strategies, our lab has developed ex vivo functional assays with small-molecule inhibitors and RNAi for the profiling of actionable targets in primary patient samples. In these assays, mononuclear cells from patient bone marrow or blood are cultured in the presence of small molecule inhibitor or RNAi libraries and cell viability is measured by colorimetric assay. To investigate targets identified in these assays, gene constructs are transfected into HEK293 T17 cells for immunoblots or transduced into mouse bone marrow cells for methylcellulose-based colony formation assays.Results: Mononuclear white blood cells from a patient with recurrent JMML were found to be sensitive to the multi-family kinase inhibitor dasatinib as well as siRNA targeting the dasatinib target tyrosine kinase non-receptor 2 (TNK2, also known as ACK1), suggesting an over-reliance on TNK2 for cell viability. TNK2 was recently demonstrated by our lab and others to regulate signaling downstream of signaling pathways including EGFR, ER, Src, and CSF3R. Based on the ex vivo functional data for this JMML specimen, the patient was placed on daily dasatinib and achieved marked improvement, which bridged the patient to an additional bone marrow transplant, resulting in disease-free survival for another year.This patient had a mutation in PTPN11 (also known as SHP2), which encodes a protein tyrosine phosphatase that is mutated in approximately 35% of JMML patients. PTPN11 is an auto-inhibited phosphatase that dephosphorylates targets in many signaling pathways including RAS-MAPK, with an overall positive effect on pathway activity and cellular proliferation. In JMML, mutations in residues of the N-terminal SH2 domain of PTPN11 result in a constitutively active form of the protein, which increases proliferation and cell survival and is linked to dysregulation of RAS/MAPK.Results of in vitro experiments demonstrate, for the first time, an interaction of TNK2 with mutant PTPN11. Co-expression of mutant-PTPN11 and TNK2 results in increased phospho-PTPN11 at two sites associated with activation of PTPN11 (Y542 and Y580), and co-immunoprecipitation experiments indicate direct interaction of TNK2 with PTPN11. These phosphorylation events coincide with significantly increased MEK/ERK signaling in the context of TNK2/mutant PTPN11 co-expression relative to expression of either gene alone. Expression of a TNK2 mutant harboring a loss-of-function mutation at a key activating residue (TNK2 Y284F) reduces PTPN11/MAPK activation, whereas expression of a gain-of-function, drug-resistant TNK2 gatekeeper mutant increases PTPN11/MAPK activation when compared to wild-type TNK2. Inhibition of TNK2 with either dasatinib or the TNK2-specific inhibitor AIM100 also reduces PTPN11/MAPK activation. Interestingly, phospho-TNK2 levels are significantly reduced in the presence of mutant-PTPN11, suggesting a feedback mechanism that has yet to be fully elucidated.Functional experiments suggest that mutant-PTPN11 and TNK2 work in a synergistic fashion to increase cell transformation and cytokine hypersensitivity. Mouse bone marrow cells transduced with both mutant PTPN11 and TNK2 have significantly more colonies in methylcellulose colony formation assays than with mutant- PTPN11 alone or with wild-type PTPN11 and TNK2. Treatment of cells in colony formation assays with either dasatinib or AIM100 inhibits colony formation, suggesting a dependence on TNK2 kinase activity for cell transformation.Conclusions: Taken together, the preliminary data suggest that PTPN11 mutations may be a functional marker for dasatinib sensitivity, and that the underlying mechanism for this sensitivity may be through the inhibition of TNK2 activity by dasatinib. Our aim is further validate the role of PTPN11 mutations as a marker for efficacy of dasatinib or other TNK2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Germline Genetic Variation in IKZF1 and Predisposition to Childhood Acute Lymphoblastic Leukemia

Background There is increasing evidence for an inherited predisposition to pediatric acute lymphoblastic leukemia (ALL). We and others have previously reported rare and highly penetrant variants in hematopoietic transcription factors (PAX5 and ETV6) and tumor suppressor genes (TP53) in both sporadic and familial ALL. IKZF1encodes the founding member of the Ikaros family of zinc finger transcription factors, and is a critical regulator of lymphoid development. IKZF1 is frequently targeted by somatic deletions and mutations in high-risk B-ALL, particularly Ph+ and Ph-like ALL, and is associated with poor outcome. IKZF1 alterations have previously been shown to result in the acquisition of stem cell-like features, overexpression of adhesion molecules causing aberrant cell-cell and cell-stroma interaction, and decreased sensitivity to tyrosine kinase inhibitors. Genome-wide association studies have also identified an association between common polymorphisms at the IKZF1locus and risk of developing ALL, however the nature and effects of germline IKZF1variation in the pathogenesis of ALL are poorly understood. In this study, we sought to comprehensively characterize germline IKZF1 genetic variation and to determine the extent to which they contribute to predisposition to ALL.Methods We recently identified a germline frameshift IKZF1 variant (D186fs) in the proband of a family with BCR-ABL1 ALL with incompletely penetrant autosomal dominant inheritance, and carriers of this variant showed varying degree of B cell deficiency. We sequenced IKZF1in germline DNA from 5,008 children with ALL (4902 B-ALL and 106 T-ALL) enrolled on ChildrenÕs Oncology Group and St. Jude ChildrenÕs Res. Hosp. frontline ALL trials. We examined each variant for its effects on transcriptional repression, DNA-binding, cellular localization, homodimerization, and leukemic cell adhesion in mouse BCR-ABL1 Arfnull B-ALL cells and/or in HEK 293T cells. All variants were assayed for their effects on cell viability and proliferation, cell-cell adhesion, and IKZF1 protein expression and localization in BCR-ABL1 Arfnull pre-B cells. Representative variants, including M31V (N-term), H163Y (DNA-binding domain), D186Tfs (familial index), M306* (truncation of C-terminus), and A434G (C-terminus) were also assayed in detail for their ability to dimerize with wild type IKZF1, bind to DNA, or dominant negative effects on transcription repressor activity in HEK293T cells. IKZF1 variants were also evaluated for inducing perturbations in cell adhesion and THY1, ITGA5, SELL expression in the mouse PreB cells, and adhesion within the bone marrow niche by ex vivo imaging of calvaria. Finally, the effects of variants on dasatinib sensitivity were assessedin vitro and in vivo.Results We identified 28 germline IKZF1variants in children with ALL, mostly in B-ALL (Figure 1). Among these variants, 3 were frameshift or nonsense resulting in truncated IKZF1 proteins. Of the remaining missense variants, 2 were located within the N-terminal DNA-binding domain, 1 in the C-terminal dimerization domain, and 22 in other parts of IKZF1 protein with clustering proximal to the C-terminal zinc fingers. In mouse BCR-ABL1 Arfnull pre-B cells, all but 4 variants (P18T, P420Q, H432Q, and M518K) variably perturbed IKZF1 function. In contrast to expression of wild-type IKZF1, which caused growth arrest, 24 of the ALL variants were tolerated; 18 caused cellular aggregation; 15 displayed cytoplasmic mislocalization; and 14 out of 20 variants analyzed had significant upregulation of the adhesion molecules THY1, ITGA5 and/or SELL that are normally repressed by IKZF1. In HEK293T cells, 3 IKZF1 truncating variants showed dramatic loss of transcription repressor activity and no longer dimerized with wildtype IKZF1. DNA-binding domain variants (R162P, H163Y) failed to repress target promoter transcription but also altered wildtype IKZF1 function in a dominant negative fashion. In comprehensively characterization of representative variants (M31V, H163Y, D186Tfs, M306X, and A434G), these variations caused cell-stroma adherence in the bone marrow niche in vivo, and significantly reduced sensitivity of leukemic cells to dasatinib in vitro and in vivo.Conclusions These results identify IKZF1 as a new ALL predisposition gene, and suggest that these germline risk variants have roles in both leukemia pathogenesis and treatment responsiveness. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Exploring Pre-B Cell Receptor Signalling As a Therapeutic Target in Acute Lymphoblastic Leukaemia

Introduction:Acute lymphoblastic leukaemia (ALL) is a clonal disorder of developing lymphocytes and is the most common malignancy in children and adolescents. While cure rates are high, treatment is associated with significant morbidity and relapsed ALL remains one of the leading causes of cancer-related deaths in children. New, less toxic therapies are clearly needed for refractory ALL.There are a number of lines of evidence to suggest that ALL cells hijack components of precursor-B cell receptor (Pre-BCR) signalling and this dependency may be amenable to therapeutic exploitation. There are a number of tyrosine kinase inhibitors (TKIs) targeting Pre-BCR signalling that are showing great promise in the clinic for other leukemia subtypes which warrant preclinical evaluation in childhood ALL.[1] These include CAL-101 (PI3K-δ inhibitor), Ibrutinib (BTK inhibitor), Fostamatinib R406 (SYK inhibitor) and Dasatinib (BCR-ABL/SRC inhibitor).Methods:TKIs were evaluated in ALL cells, including cell lines (PreB 697 and its glucocorticoid resistant descendant, R3F9; Nalm-6 and Reh) and 25 patient derived xenograft samples (PDX) from 12 predominantly high risk/relapse children ALLs. Resazurin was used to assess cell viability. Flow cytometry was used to detect Pre-BCR expression (µHc, Vpreb and λ5) and functionality using a Calcium flux assay. Phospho-flow cytometry was performed to monitor constitutive phosphorylation and response to Pre-BCR activation and to assess pharmacodynamic drug action (p-AKT, p-BLNK, p-BTK, p-SYK, p-ERK, and p-PLC-ϒ2). GILZ expression was measured by RQ-PCR. Cell cycle and apoptosis were determined by flow cytometry using Propidium Iodide and Annexin V staining.Results:ALL cell lines were resistant to CAL-101 (mean GI50 52.08 µM, range 25 µM-77.83 µM) and Ibrutinib (mean GI50 15.9 µM, range 11.47 µM-18.3 µM). However, modest sensitivity was seen to R406 (mean GI50 4.32 µM, range 2.88 µM-5.83 µM) and Dasatinib (mean GI50 5.33 µM, range 2.45 µM-12.5 µM). CAL-101 and Dasatinib were shown to be cytostatic, causing G1 arrest but no significant apoptosis, while Ibrutinib and R406 were associated with cell cycle arrest and significant apoptosis after 72 hours incubation with GI50 concentrations (16.81±1.71 % and 31.34±5.78 % apoptosis, respectively). Pre-B receptor positive cells were more sensitive to Dasatinib and Fostamatinib. Pharmacodynamic assessment using Phospho-flow cytometry and Western blotting showed inhibition of the relevant targets at the GI50 concentrations. PDX ALL cells were generally more sensitive than the cell lines; CAL-101 (mean GI50 25.56 µM, range 76 nM-100 µM; 2 out of 12 patient samples <2µM); Ibrutinib (mean GI50 14.23 µM, range 490 nM-100 µM; 3 out of 12 patient samples <5µM); R406 (mean GI50 11.52 µM and range 56 nM-25 µM, 4 out of 12 patient samples <4µM); Dasatinib (mean GI50 25.56 µM, range 76 nM-100 µM; 3 out of 12 patient samples <0.5µM). ALLs sensitive to Dasatinib were Ph+ or Pre-BCR positive and the latter were also sensitive to Fostamatinib.Synergism was seen after co-treatment of TKIs with the glucocorticoid (GC), Dexamethasone (CAL-101 CI mean 0.7, range 0.056-1.34; Ibrutinib CI mean 0.71, range 0.41-0.97; R406 CI mean 0.27, range 0.1-0.6 and Dasatinib CI mean 0.63, range 0.14-1.29). No synergism was observed in the glucocorticoid receptor negative, Reh cell line. For R406 and Dasatinib, co- exposure was strongly synergistic and was associated with increased apoptosis in PreB 697 and the GC resistant lines, Nalm-6 and R3F9. Synergism was associated with a significant increase in expression of the GR target, GILZ and an enhanced downregulation of R406 and Dasatinib targets (p-SYK for R406 and p-BTK, p-SYK for Dasatinib).Conclusion:We have identified significant sensitivity of TKIs impacting on Pre-BCR signalling in ALL cells at clinically relevant concentrations; Pre-BCR positive ALLs were associated with Dasatinib and Fostamatinib sensitivity; Pre-BCR negative ALL cells were also sensitive to some TKIs, although predictive biomarkers remain to be established. Marked synergism was observed in combination with dexamethasone, even in GC resistant cells. In vivo preclinical confirmation of these data may offer new therapies for refractory ALL.1. Young, R.M. and L.M. Staudt, Targeting pathological B cell receptor signalling in lymphoid malignancies. Nature Reviews Drug Discovery, 2013. 12(3): p. 229-243. DisclosuresNo relevant conflicts of interest to declare.

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.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib.

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR.

A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer.

Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity.

Abstract 399: Nanoparticle silencing of β1 and β3 integrins augments Dasatinib sensitivity

Abstract Triple negative breast cancers (TNBCs) are an aggressive subcategory of breast cancers that currently lack FDA-approved targeted therapies. Previous studies have i) further subcategorized TNBCs into 5 distinct subtypes, each with a preferential response to specific chemotherapies and ii) identified beta1 and beta3 integrins to be key mediators of breast cancer EMT and metastasis. We surveyed beta1 and beta3 integrin expression in the 5 subtypes of TNBC with and without treatment of their proposed chemotherapies by western blot analysis. Using nanoparticles, we delivered beta1 and beta3 integrin-specific siRNAs to silence the expression of both integrins either with or without treatment of the proposed chemotherapies, and evaluated the effect of these treatment regimens on EMT, invasion, and 3D outgrowth. We demonstrated that beta1 and beta3 integrins are robustly expressed in the Mesenchymal Stem-Like, Mesenchymal, and Basal-Like2 subtypes of TNBCs. Furthermore, we observed elevated expression of beta1 integrin upon Dasatinib treatment of the Mesenchymal Stem-Like subtype. Lastly, we determined that delivery of beta1 and beta3 integrin siRNAs sensitized breast cancer cells to Dasatinib treatment. Collectively, our data suggests that delivery of beta1 and beta3 integrin-specific siRNAs in combination with Dasatinib treatment efficiently eliminates the aggressive characteristics of Mesenchymal Stem-Like breast cancer cell lines. Citation Format: Jenny G. Parvani, Vinson Chu, Sarah Roelle, Zheng-Rong Lu, William Schiemann. Nanoparticle silencing of β1 and β3 integrins augments Dasatinib sensitivity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 399. doi:10.1158/1538-7445.AM2015-399

Abstract IA10: Kinase-impaired BRAF mutations as predictors of resistance and sensitivity

Abstract Non-small cell lung cancer (NSCLC) is a common and rapidly fatal cancer for which targeted therapies have been markedly effective in only about 15% of patients - specifically those with EGFR mutations or EML4-ALK translocations. During a clinical trial of the multi-targeted tyrosine kinase inhibitor dasatinib we identified a patient with stage IV NSCLC who harbored a novel BRAF mutation (Y472C), had a near complete radiographic response to the multitargeted kinase inhibitor dasatinib as sole therapy and remained without active cancer 7 years following treatment. We discovered that Y472CBRAF is a kinase-inactivating BRAF mutation (KIBRAF). BRAF mutations can result in increased or decreased BRAF kinase activity, as well as kinase-neutral mutations, and occur in 8% of NSCLC and many other tumor types. We found that NSCLC cells that harbor KIBRAF undergo senescence when exposed to the dasatinib whereas NSCLC patients and cell lines with wild type BRAF are resistant to dasatinib. Transfection of other NSCLC cells with KIBRAF mutations also increased their dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. NSCLC cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation. Currently there are no well-defined, canonical pathways that explain the observed dasatinib-induced senescence in NSCLC cells with KIBRAF. Dasatinib inhibits the activity of 40 distinct kinase targets and can induce BRAF-CRAF dimerization and CRAF activation in cells with activated RAS or KIBRAF mutations. To investigate the mechanism leading to senescence we performed reverse phase protein arrays to simultaneously examine the expression of 137 proteins and phosphoproteins and gene expression arrays. This approach was limited by the existence of only two NSCLC cell lines with KIBRAF but nonetheless we determined that dasatinib induced DNA damage and subsequently activated DNA repair pathways and the HIPPO signaling pathway only in NSCLC cells with KIBRAF. We are currently conducting a clinical trial for NSCLC patients with KIBRAF mutations to determine the efficacy of dasatinib in these patients prospectively. Citation Format: Faye Johnson, Banibrata Sen, Tuhina Mazumdar, Shaohua Peng, Humam Kadara, Lixia Diao, Lauren Byers. Kinase-impaired BRAF mutations as predictors of resistance and sensitivity. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr IA10.

Dasatinib Shows Therapeutic Potential in the Murine Xenograft Model for TCF3 rearranged Acute Lymphoblastic Leukemia

Transcription Factor 3 (TCF3) rearrangements are a recurring chromosomal abnormality in B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) occurring in approximately five percent of pediatric ALL. Historically, the majority of these patients carried a poor prognosis, but advances with more intensive cytotoxic chemotherapy have improved the survival rate while exposing patients to increased short and long-term toxicities. Two genetic rearrangements produce the chimeric transcription factors, TCF3-PBX1 t(1;19)(q23;p13) and a much rarer TCF3-HLF t(17;19)(q22;p13). Sadly, TCF3-HLF remains an extremely difficult disease to treat with few, if any known survivors. Although it is unknown how these translocations lead directly to disease, it is established that they do result in diseases arrested in a later stage of B-cell differentiation and pre-B cell receptor (pre-BCR) dependence. Recently, we highlighted the concept of targeting the pre-BCR pathway for therapeutic potential using dasatinib (Sprycel). Here, we further examine dasatinib effectiveness in the murine xenograft model for TCF3-rearranged ALL. Methods: Primary patient samples were obtained with written informed consent approved by the Institutional Review Board of Oregon Health and Science University and processed. Mononuclear cells were separated by Ficoll and exposed to increasing concentrations of dasatinib. Inhibitory Concentration of fifty-percent viability (IC50) was calculated for each sample. The median IC50 for over four hundred acute leukemic samples interrogated by this assay was calculated to approximately 100nM. Samples with IC50 values below 30nM were deemed hypersensitive to dasatinib. For xenografts, frozen viable primary patient samples were thawed and grafted via tail-vein into NOD/SCID/IL-2rgnull(NSG) mice 24 hours after sub-lethal irradiation with 200 cGy. Upon engraftment, and in vivo expansion, animals were euthanized and leukemic cells recovered from the spleen were then injected in secondary recipients. One week after injection the mice were divided into two groups and treated by oral gavage with dasatinib at 50mg/kg/dose daily or citrate control for 5 days per week. Treatment continued until the day of sacrifice (4-20 weeks). Peripheral blood engraftment was monitored weekly starting on week 3 by flow cytometry analysis using anti-human CD19 and CD45 (hCD19-APC, hCD45-FITC) versus anti-murine CD45 (mCD45-PerCP-Cy5.5). Flow cytometric data was analyzed using FACS/AriaIII. Results: Screening over one hundred BCP-ALL samples identified that approximately ten percent of these samples show hypersensitivity to dasatinib. TCF3-rearranged ALL and BCR-ABL1 ALL had a majority of samples with IC50’s less than 10nM. Throughout all known subsets of ALL except ETV6-RUNX1, there also appeared to be individual samples that have IC50 values less than 30nM, suggesting significant sensitivity to this drug. Of these, three individual TCF3-rearranged ALL samples were identified and xenografted into NSG mice, expanded and injected into secondary recipients. All dasatinib treated cohorts showed significantly less leukemic peripheral blood chimerisms as compared to their vehicle control counterparts. Further, in vitro treatment of xenografted cells with dasatinib indicated inhibition of the pre-BCR by decrease in pan-phospho-SRC. Intriguingly, dasatinib did not completely abolish disease in all TCF3-rearranged ALL, suggesting other important mechanisms for cell viability. Conclusions: These studies show in vivo therapeutic benefits of dasatinib as treatment for TCF3-rearranged ALL, and open the possibility of adding this drug to their treatment. Further studies are underway to address the mechanisms of dasatinib sensitivity of other subsets of ALL identified in our screen in hopes of adding targeted therapies to their treatment. [Display omitted] DisclosuresDruker:Molecular MD: Consultancy, Equity Ownership, Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. Other; Bristol-Myers Squibb: Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other. Off Label Use: Dasatinib use as potential therapy in ALL.

319 Genomic profiling of uterine leiomyosarcomas reveal frequent alterations in Akt/mammalian target of rapamycin (mTOR) pathway genes and other actionable genomic abnormalities linked to targeted therapies

Based on our current understanding of ARID1A function, it is difficult to predict a therapeutic strategy for these tumours. Therefore we adopted an unbiased approach using integrated functional and molecular profiling of ARID1A mutant and wild-type tumour cell models. Methods: We profiled two parallel systems; a panel of ARID1A mutant and wild-type OCCC cell lines models and an isogenic pair of ARID1A proficient and deficient HCT116 cell lines, using high throughput screening with chemical and siRNA libraries. This has been integrated with molecular profiling data generated from exome and RNA sequencing to aid the discovery of novel targets. Results: Using functional screens we have identified critical genetic dependencies and potential therapeutics in a series of clear cell ovarian cancer models. Specifically, we have established that dasatinib is selective for OCCC models with loss of function ARID1A mutations both in vitro and in vivo. Dasatinib is not only selective for ARID1A mutant OCCC models, but experimental induction of ARID1A deficiency drives dasatinib sensitivity in ARID1A wild-type OCCC models as well as in isogenic cell systems (both mouse and human) where ARID1A has been rendered dysfunctional by gene targeting. Conclusions: These results provide a framework for the discovery of therapeutic targets for clear cell ovarian cancer characterised by ARID1A mutations. Using an unbiased drug and siRNA sensitivity screening approach, we have identified dasatinib as a candidate therapeutic approach for OCCC. This Dasatinib/ARID1A synthetic lethal approach can be observed both in in vitro and in vivo and suggests that dasatinib should be assessed in clinical trials in OCCC patients.