Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL) remains a challenge. Although the addition of targeted tyrosine kinase inhibitors (TKIs) to standard cytotoxic therapy has greatly improved upfront treatment, treatment-related morbidity and mortality remain high. TKI monotherapy provides only temporary responses and renders patients susceptible to the development of TKI resistance. Thus, identifying agents that could enhance the activity of TKIs is urgently needed. Recently, a selective inhibitor of B cell lymphoma 2 (BCL-2), ABT-199 (venetoclax), has shown impressive activity against hematologic malignancies. We demonstrate that the combination of TKIs with venetoclax is highly synergistic in vitro, decreasing cell viability and inducing apoptosis in Ph(+)ALL. Furthermore, the multikinase inhibitors dasatinib and ponatinib appear to have the added advantage of inducing Lck/Yes novel tyrosine kinase (LYN)-mediated proapoptotic BCL-2-like protein 11 (BIM) expression and inhibiting up-regulation of antiapoptotic myeloid cell leukemia 1 (MCL-1), thereby potentially overcoming the development of venetoclax resistance. Evaluation of the dasatinib-venetoclax combination for the treatment of primary Ph(+)ALL patient samples in xenografted immunodeficient mice confirmed the tolerability of this drug combination and demonstrated its superior antileukemic efficacy compared to either agent alone. These data suggest that the combination of dasatinib and venetoclax has the potential to improve the treatment of Ph(+)ALL and should be further evaluated for patient care.

BackgroundNovel-targeted therapies are in rapid development for the treatment of acute lymphoblastic leukemia (ALL) to overcome resistance and decrease toxicity. Survivin, a member of the inhibitor of apoptosis gene family and chromosome passenger complex, is critical in a variety of human cancers, including ALL. A well-established suppressor of survivin has been the small molecule, YM155. Reports are identifying other mechanisms of action for YM155. Therefore, we sought to investigate the mode of action and role of YM155 for therapeutic use in the context of ALL.MethodsPrimary ALL samples and ALL cell lines were interrogated with YM155 to identify drug sensitivity. Ph+ALL harboring the BCR-ABL1 oncogene were tested for any interaction with YM155 and the multi-kinase inhibitor dasatinib. Representative ALL cell lines were tested to identify the response to YM155 using standard biochemical assays as well as RNA expression and phosphorylation arrays.ResultsALL samples exhibited significant sensitivity to YM155, and an additive response was observed with dasatinib in the setting of Ph+ALL. ALL cells were more sensitive to YM155 during S phase during DNA replication. YM155 activates the DNA damage pathway leading to phosphorylation of Chk2 and H2AX. Interestingly, screening of primary patient samples identified unique and exquisite YM155 sensitivity in some but not all ALL specimens.ConclusionThese results are the first to have screened a large number of primary patient leukemic samples to identify individual variations of response to YM155. Our studies further support that YM155 in ALL induces DNA damage leading to S phase arrest. Finally, only subsets of ALL have exquisite sensitivity to YM155 presumably through both suppression of survivin expression and activation of the DNA damage pathway underscoring its potential for therapeutic development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0132-6) contains supplementary material, which is available to authorized users.

DLBCL accounts for >10,000 deaths in the U.S. annually. Despite efficacy of upfront chemo-immunotherapy, up to 50% of patients die within 5 years. While inhibitors of B-cell receptor signaling transformed the therapeutic paradigm in indolent lymphomas, they show poor activity in DLBCL, which thus represents an unmet clinical need. MYC oncogene, a transcription factor which regulates cell proliferation and growth, is a key driver in lymphomagenesis. Myc overexpression is associated with chemoresistance in NHL, while "double-hit" lymphomas characterized by MYC and BCL2 rearrangement are most aggressive. Myc proteins have extraordinarily short half-life. To regulate transcription, Myc recruits pTEFb, an elongation factor whose catalytic core comprises CDK9/cyclin T complexes. Hence, Myc expression and function may be susceptible to CDK9 inhibition. Non-specific inhibitors of multiple CDKs have shown promise in B-cell malignancies, where their pro-apoptotic effect has been attributed to a reduction in transcription and downmodulation of short lived pro-survival proteins, e.g. MCL1. However, such inhibitors lack a therapeutic window or a refined mechanism of action. Here we selectively targeted CDK9 in a pre-clinical study in DLBCL and demonstrate that this therapeutic approach disrupts Myc oncogenic function. Experiments were conducted in activated B-cell(ABC)-type (OCI-LY3, U2932, NuDUL-1) and germinal center(GC)-type (OCI-LY18/19, SUDHL4/6/10/16, VAL) DLBCL cell lines and in primary lymph node-derived B-cells from patients with refractory DLBCL. Tumor xenografts were established in NSG mice. We used AZ5576, a novel selective CDK9 inhibitor (Astra Zeneca), pan-CDK inhibitors (flavopiridol, dinaciclib) and genetic knockdown of CDK9. Treatment with AZ5576 promoted rapid apoptosis induction (within 2 hours) in DLBCL cell lines and primary tumor cells, as evident by PARP cleavage. Quantification of Annexin V+ cells after 24 hour drug exposure rendered an IC50 of 300-500 nM in DLBCL cell lines independent of cell of origin and 100 nM in primary DLBCL cells. Loss of RNA polymerase phosphorylation at Ser2 residue, a CDK9-dependent site, preceded apoptosis. Contrary to pan-CDK inhibitors, AZ-5576 did not inhibit CDKs 2, 4/6, and 7 (as measured by pRb and pRNApol [Ser5]), confirming its selective effect. CDK9 inhibition resulted in rapid (within 1 hour) and dose-dependent downregulation of Myc and MCL1 mRNA and protein in DLBCL cell lines and primary cells. Cell cycle profiling demonstrated that treatment with AZ-5576 led to S phase reduction, consistent with loss of Myc, while pan-CDK inhibitors downmodulated S/G2/M, reflecting inhibition of multiple CDKs. Cyclohexamide chase experiments demonstrated enhanced degradation of Myc in DLBCL cells treated with AZ-5576. Myc protein degradation is tightly regulated: while phosphorylation at Ser62 enhances Myc stability, MycT58 is primed for degradation. CDK9 inhibition decreased pMycS62 prior to apoptosis induction in DLBCL cell lines. Raji cells, which carry a stabilizing MycT58A mutation, were less sensitive to AZ-5576. This suggests that CDK9 may govern Myc protein turnover, thus regulating its expression through multiple mechanisms. Overexpression of Myc was associated with enhanced sensitivity to CDK9 inhibition. Treatment with 300 nM AZ-5576 for 24 h induced apoptosis of 35-70% of Myc-high cells (e.g., U-2932, VAL), compared with 10-20% of Myc-low cells (e.g., OCI-LY3, SU-DHL10). CDK9 inhibition did not disrupt Myc-CDK9 complex formation. Despite this, it abrogated Myc transcriptional activity in a functional reporter assay. Meanwhile, sensitivity to AZ-5576 did not correlate with expression of MCL1, BCL2 or BCLX. Our findings were confirmed in a mouse xenograft model. VAL cells were inoculated subcutaneously and treatment with AZ-5576 (30 mg/kg orally twice weekly) or vehicle control began when tumors reached 10 mm in size. Treatment led to reduced tumor progression (Figure), prolonged survival, decreased cell proliferation (Ki-67), enhanced apoptosis (Annexin V), and reduction in Myc total and p-S62 levels and MCL1. Thus, our data suggests that targeting CDK9 is a promising therapeutic strategy poised to disrupt Myc oncogenic activity in DLBCL and provides rationale for clinical development of AZ-5576. Figure 1 Figure 1. Disclosures Chen: Seattle Genetics: Consultancy. Drew:Astra Zeneca: Employment. Danilov:ImmunoGen: Consultancy; Pharmacyclics: Consultancy; Astra Zeneca: Research Funding; Prime Oncology: Honoraria; Dava Oncology: Honoraria; GIlead Sciences: Research Funding; Takeda: Research Funding.

Introduction: Treatment of adult Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) remains a challenge. While the addition of the targeted tyrosine kinase inhibitors (TKI) to standard cytotoxic therapy has greatly improved upfront treatment, treatment related mortality in older adults remains high. A novel induction regimen combines the targeted dual Abl/Src TKI Dasatinib (Sprycel, BMS) with a corticosteroid. After the first 21 days of induction the corticosteroids are tapered due to significant toxicities, particularly in older adults. Unfortunately, remaining on TKI monotherapy renders patients susceptible to the development of TKI resistance and thus identifying targeted agents that could enhance the activity of TKIs is urgently needed. Recently a novel and selective inhibitor of BCL-2, ABT-199 (Venetoclax, AbbVie) has shown impressive activity against other lymphoid malignancies including CLL and NHL. Here we describe the pre-clinical and in vivo efficacy of ABT-199 in combination with dasatinib in Ph+ ALL and propose its potential use in future clinical trials. Methods: Drug efficacy in vitro was determined using the Ph+ ALL cell line SupB15, primary Ph+ ALL sample (12-149), the dasatinib sensitive Pre-B ALL cell line RCH and the CML cell line K562. Cells were treated with dasatinib, ABT199 or in combination for 72 hours. Cell viability was assessed with the colorimetric MTS assay and apoptosis was assessed with annexin V staining. Expression of the BCL family proteins BCL-2 and MCL-1 were assessed via immunoblot. Immunodeficient NSG mice were injected with 12-149, then one week later treated with vehicle, 5 mg/kg dasatinib, 5 mg/kg ABT-199, or the combination daily for 5 days each week. Peripheral blood was obtained every 1-2 weeks to assess for engraftment as defined by the presence of >10% human CD45+ cells in the peripheral blood. Once engrafted, mice were euthanized and examined. Mononuclear cells were extracted and assessed for BCL2 and MCL1 expression. Statistical methods were performed using Calcusyn and PRISM. Results: Susceptibility to BCL2 inhibition: Of the dasatinib sensitive cells tested, SupB15 and 12-149 cells were susceptible to ABT-199 while RCH and K562 cells were not. The ALL cells expressed BCL-2 while the CML cell line expressed BCLx. SupB15 expressed low levels of the antiapoptotic protein MCL1 while RCH cells had relatively higher levels. siRNA of MCL-1 rendered the RCH cells sensitive to inhibition by ABT-199. In SupB15 cells, treatment with ABT-199 alone led to upregulation of MCL-1 at 24h which was prevented by the combination of dasatinib + ABT199. Synergy in Ph+ ALL: The calculated IC50 of dasatinib and ABT199 in SupB15 were 8.8nM and 5.9nM, respectively. The IC50 of equimolar combination was 0.42nM, and synergistic with combination index (CI) values between 0.15 and 0.49. Primary Ph+ ALL xenograft cells showed a similar pattern of synergy to the dasatinib + ABT199 combination. Combination treatment also greatly increased apoptosis as measured by Annexin V staining. Xenograft Studies: Animals were treated with a ten-fold lower dose of dasatinib and ABT199 from prior published data. There was no significant difference in time to engraftment or disease burden between vehicle or single agent ABT-199. In contrast, less than one half of the animals treated with dasatinib engrafted by 90 days while none of the animals treated with both dasatinib and ABT-199 engrafted. Most intriguing was the decrease in disease burden as measured by splenic size in the combination group compared to all other groups (P<0.0001, one-way ANOVA). Analysis of BCL-2 family proteins from mononuclear cells isolated from untreated animals confirmed upregulation of BCL-2 and relatively low levels of MCL-1. Animals treated with ABT-199 had greatly upregulated levels of MCL-1, while those treated with dasatinib or the combination did not. Conclusions: The combination of ABT-199 with dasatinib synergistically targets Ph+ ALL cells both in vitro and in vivo, laying the foundation for further evaluation in vivo for adult Ph+ ALL. As demonstrated by others, malignancies that are particularly susceptible to BCL targeting are those which display high BCL-2 expression and a low MCL-1: BCL-2 ratio. Combined targeted therapies may offer the potential for greater and longer responses without the morbidity associated with cytotoxic chemotherapy, particularly in older adults. Disclosures Tyner: Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Array Biopharma: Research Funding; Constellation Pharmaceuticals: Research Funding. Druker:Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Novartis Pharmaceuticals: Research Funding; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy.

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. Disclosures Tyner: 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.