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Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome–positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.

Recent studies have revealed that p27, a nuclear cyclin-dependent kinase (Cdk) inhibitor and tumor suppressor, can acquire oncogenic activities upon mislocalization to the cytoplasm. To understand how these antagonistic activities influence oncogenesis, we dissected the nuclear and cytoplasmic functions of p27 in chronic myeloid leukemia (CML), a well-characterized malignancy caused by the BCR-ABL1 tyrosine kinase. p27 is predominantly cytoplasmic in CML and nuclear in normal cells. BCR-ABL1 regulates nuclear and cytoplasmic p27 abundance by kinase-dependent and -independent mechanisms, respectively. p27 knockdown in CML cell lines with predominantly cytoplasmic p27 induces apoptosis, consistent with a leukemogenic role of cytoplasmic p27. Accordingly, a p27 mutant (p27(CK-)) devoid of Cdk inhibitory nuclear functions enhances leukemogenesis in a murine CML model compared with complete absence of p27. In contrast, p27 mutations that enhance its stability (p27(T187A)) or nuclear retention (p27(S10A)) attenuate leukemogenesis over wild-type p27, validating the tumor-suppressor function of nuclear p27 in CML. We conclude that BCR-ABL1 kinase-dependent and -independent mechanisms convert p27 from a nuclear tumor suppressor to a cytoplasmic oncogene. These findings suggest that cytoplasmic mislocalization of p27 despite BCR-ABL1 inhibition by tyrosine kinase inhibitors may contribute to drug resistance, and effective therapeutic strategies to stabilize nuclear p27 must also prevent cytoplasmic mislocalization.

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2V617F expressing cells in MPN. We show that JAK2V617F kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2V617F allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2V617F-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2V617F expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2V617F-positive MPN. Altogether our data are consistent with a model where JAK2V617F promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.

Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naive subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both 'native' CD4+ CD25+ and 'inducible' CD4+ CD25- peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.

Chronic beryllium disease (CBD) is characterized pathologically by granulomatous inflammation in the lung, composed of a large core of epithelioid cells surrounded by a dense shell of CD4+ T cells. Using beryllium-specific CD4+ T cell lines derived from the bronchoalveolar lavage (BAL) fluid of CBD patients, we show that purified CD4+ T cells produced significant amounts of IFN-gamma and TNF-alpha upon exposure to beryllium in the absence of antigen-presenting cells (APC). However, unlike BAL T cells stimulated by beryllium in the presence of APC, self-presentation by BAL T cells did not induce detectable IL-2 production, and in its absence these activated T cells die from programmed cell death. Resting BAL CD4+ T cells constitutively express high levels of HLA-DP, lymphocyte function-associated antigen 1 (LFA-1) and ICAM-3. When stimulated with beryllium/APC, the adhesion molecule ICAM-1 was up-regulated, as well as several costimulation molecules including CD28, OX-40 (CD134), 4-1-BB (CD137) and B7-1 (CD80). Notably, CD28 was not up-regulated during self-presentation by BAL T cells, and these cells do not express OX-40L, suggesting that lack of appropriate costimulation was responsible for programmed cell death observed upon beryllium self-presentation. Restricting anti-MHC class II mAb completely eliminated beryllium-induced T cell proliferation during self-presentation and significantly reduced IFN-gamma and TNF-alpha production. Our data demonstrate for the first time that self-presentation by BAL T cells in response to beryllium can occur ex vivo, in the absence of professional APC, with a specific dependence on T cell-expressed MHC class II molecules and exogenous IL-2 for survival.

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. Figure 1 Figure 1. Disclosures Druker: 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.

Abstract 3504 The imatinib paradigm established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKIs). However, once-daily dasatinib (serum half-life: 3–5 h) is clinically effective despite only transient BCR-ABL inhibition, opening an opportunity for in-depth study of the mechanistic requirements for ABL TKI-induced CML cell death. Apoptosis commitment after potent, transient target inhibition is observed with ABL TKIs in vitro (Blood, 114, 2009, 3459–63), although variations in required TKI concentrations relative to their activity against BCR-ABL kinase suggest involvement of previously unrecognized factors. The “oncogenic shock” concept holds that temporary disruption of BCR-ABL-mediated prosurvival and proapoptotic signaling sets up a kinetic imbalance in favor of apoptosis. We have undertaken a comprehensive mechanistic exploration of this issue, wherein CML cells were transiently exposed to the ABL TKIs imatinib (50 and 500 nM), nilotinib (50 and 500 nM), dasatinib (10 and 100 nM), and ponatinib (AP24534; 10 and 100 nM) and then investigated with respect to pathways critical to drug efficacy and intracellular residence time. Cellular studies utilized multi-parameter intracellular FACS and immunoblot analysis, liquid chromatography-mass spectrometry, and high-throughput real-time qPCR expression analysis. Corresponding biochemical studies to determine ABL kinase/inhibitor dissociation parameters were also performed. All four ABL TKIs tested were capable of triggering apoptosis following transient exposure, although nilotinib and imatinib (which feature much narrower kinase target profiles than dasatinib and ponatinib) did so only at high concentrations. In contrast to potent, transient inhibition of BCR-ABL being necessary and sufficient for commitment of CML cells to apoptosis, we found that apoptosis could be reversed under conditions involving extensive additional TKI washout protocols. Consistent with the best indicator of apoptosis induction in our experiments being incomplete restoration of BCR-ABL signaling activity to pre-treatment levels, in all cases for which apoptosis commitment was irreversible, we identified a small, functionally important pool of intracellular TKI after washout of drug from the media. This property correlated with results of ABL kinase/inhibitor dissociation studies. For example, we found that ponatinib is a tight-binding inhibitor with a remarkably slow off-rate (t1/2 >95 h). Transient exposure to ponatinib followed by thorough washout committed CML cells to apoptosis despite very low intracellular concentrations that did not completely inhibit BCR-ABL. Based on these findings, we explored the possibility that effective TKIs are inhibiting additional targets involved in committing cells to an apoptotic fate, and used high-throughput qPCR assays to identify a preliminary profile of 30 apoptosis-related genes differentially expressed in TKI-treatment conditions that do and do not irrevocably commit CML cells to apoptosis. Taken together, our findings reveal that even slightly attenuated restoration of BCR-ABL signaling correlates with apoptosis commitment and that cryptic cellular retention of ABL TKIs is important in mediating this effect, potentially via sustained low-level inhibition of auxiliary targets. By extension, monitoring intracellular drug levels by LC-MS may be informative, especially for short serum half-life kinase inhibitors such as dasatinib. These studies further establish and refine the guiding principles of commitment of CML cells to apoptosis and improve our ability to design kinase inhibitors for CML and other malignancies. Disclosures: Riddle: Life Technologies Corporation: Employment, Equity Ownership. Apgar:BD Biosciences: Employment. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential COI has been reviewed and managed by the OHSU COI in Research Committee & Integrity Program Oversight Council.

Abstract 512 Background: BCR-ABL promotes cell cycle progression by interfering with the regulatory functions of p27, a cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. We have previously shown that BCR-ABL kinase activity promotes degradation of nuclear p27 (Agarwal, A. et al. Blood 2008). Additionally, in primary CML cells, p27 is mislocalized to the cytoplasm, thereby relieving Cdks from p27 inhibition. Results from studies of solid tumors show that cytoplasmic p27 can actively contribute to oncogenesis, raising the question of whether cytoplasmic p27 in CML cells may actively promote leukemogenesis rather than merely compromise Cdk inhibition. We hypothesize that BCR-ABL disrupts p27 function in a dual manner by reducing nuclear p27, where p27 normally serves as a tumor suppressor, and by increasing cytoplasmic p27, where it might have oncogenic activity. Experimental Approach and Results: Immunoblotting of nuclear and cytoplasmic lysates of CD34+ cells from 11 CML patients revealed that p27 localization is predominantly cytoplasmic in the majority of patients (10/11; 91%) irrespective of disease phase, while p27 was mostly nuclear in normal controls. Similar results were obtained by immunofluorescence microscopy. Imatinib treatment increased nuclear p27 suggesting that nuclear p27 levels are regulated by BCR-ABL kinase activity. However, imatinib does not alter cytoplasmic p27 levels, suggesting that cytoplasmic mislocalization of p27 is a kinase-independent effect of BCR-ABL. Kinase-independent regulation of cytoplasmic p27 localization was also tested by immunofluorescence microscopy of p27−/− MEFs engineered to express active or kinase-dead BCR-ABL in combination with wild-type p27. In these cells cytoplasmic p27 abundance was increased both by kinase-active or kinase-dead BCR-ABL as compared to the vector control. To interrogate the role of p27 in vivo we retrovirally transduced p27+/+ or p27−/− bone marrow with BCR-ABL-GFP retrovirus and sorted Lin-/c-Kit+/Sca-I+ cells by FACS, allowing for injection of exactly matched numbers of BCR-ABL-expressing GFP+ cells (5000/animal). Median survival was significantly reduced for recipients of p27−/− marrow as compared to p27+/+ controls (34 days vs. 93 days p Conclusions: These results provide in vivo evidence that p27 has genetically separable dual roles in CML as both a nuclear tumor suppressor and cytoplasmic oncogene. A kinase-independent activity of BCR-ABL contributes to leukemogenesis through aberrant p27 localization to the cytoplasm. This oncogene activity is independent from the kinase-dependent degradation of nuclear p27. We speculate that the inability of tyrosine kinase inhibitors to reverse cytoplasmic p27 mislocalization may contribute to disease persistence despite effective inhibition of BCR-ABL kinase activity. Disclosures: Deininger: Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; genzyme: Research Funding.

Abstract 3276 Poster Board III-1 Background: Several studies have indicated that BCR-ABL causes cell cycle defects by interfering with the cell cycle regulatory functions of p27, a Cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. Studies in BCR-ABL positive cell lines have shown that BCR-ABL promotes proteasomal degradation of p27 in a pathway that involves the SCFSKP2 ubiquitin ligase, while cytoplasmic mislocalization has been described in primary CML cells. It has been suggested that the principal effect of this cytoplasmic mislocalization is to remove p27 from the nucleus, thereby relieving Cdks from p27 inhibition. However, recent studies have shown that a p27 mutant (p27CK-), that cannot bind to Cdks or Cyclins, actively contributes to oncogenesis. This raises the question as to whether cytoplasmic mislocalization of p27 in CML cells may in fact promote leukemogenesis rather than merely compromise Cdk inhibition. We therefore hypothesized that the net contribution of p27 in CML is to promote leukemogenesis due to the oncogenic activity of cytoplasmic p27. Experimental approach and results: We determined p27 localization in BCR-ABL positive cell lines and CD34+ progenitor cells from newly diagnosed chronic phase CML patients (N=7) and from CML patients in blast crisis (N=2) by immunoblotting of nuclear and cytoplasmic cellular fractions. We found that p27 is predominantly cytoplasmic in most CML cell lines and in CD34+ cells from 8/9 (89%) patient samples, including patients in blastic phase. Cytoplasmic localization of p27 in CD34+ cells from CML patients was also confirmed by immunofluorescence analysis. Further, we observed that inhibition of BCR-ABL kinase by imatinib, an Abl kinase inhibitor increased nuclear p27 in all cell lines tested and in 4/9 patient samples (3/7 chronic phase and 1/2 blastic phase samples). However, we did not observe a substantial change in the cytoplasmic p27 levels. Similar results were obtained in Ba/F3 and 32D murine hematopoietic cell lines expressing BCR-ABL when compared with the respective parental cells. Further, SKP2 was up-regulated in CD34+ cell from CML patients as compared to the normal patients consistent withSKP2 mediated down-regulation of nuclear p27. These data suggest that nuclear but not cytoplasmic p27 levels are predominantly regulated by BCR-ABL kinase activity. To test whether p27 is crucial for BCR-ABL-driven leukemia, we compared leukemogenesis between recipients of BCR-ABL transduced p27+/+ and p27-/- bone marrow. Mice transplanted with BCR-ABL infected p27-/- marrow had significantly longer median survival (70 days, range 48-150 days) compared to recipients of p27+/+ marrow (37 days, range 14-56 days) (p=0.0123). To exclude that this difference was related to the differences in homing and engraftment capabilities of p27+/+ and p27-/- bone marrow cells, we compared short term homing and long term engraftment of p27+/+ and p27-/- bone marrow cells transplanted into wild-type recipients and found no differences. These data suggest that the net contribution of p27 to BCR-ABL-mediated leukemogenesis is positive. Further, to investigate the contribution of nuclear p27 to leukemogenesis, we utilized marrow from p27S10A mice in the murine CML model. In p27S10A mice, p27 is nuclear to to abrogation of the phosphorylation site implicated in nuclear export. We injected BCR-ABL transduced bone marrow cells of p27S10A and p27+/+ mice into wild-type recipients and compared the disease progression. We observed that mice transplanted with BCR-ABL infected p27S10A marrow had significantly longer median survival (28 days, range 23-79 days) compared to the recipients of p27+/+ marrow (23 days, range 21-38 days) (p=0.0139). This data is consistent with nuclear tumor suppressor function of p27. Combined with the data above, this suggests that cytoplasmic p27 promotes BCR-ABL mediated leukemogenesis. Conclusions: Our data suggest that though nuclear p27 functions as a tumor suppressor, the net contribution of p27 in CML might be oncogenic due to an oncogenic role of the increased cytoplasmic p27. Restoring nuclear p27 or reducing cytoplasmic p27 may be therapeutically useful in malignancies with low nuclear and high cytoplasmic p27 expression. Disclosures: Druker: OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Deininger:Genzyme: Research Funding; BMS: Consultancy; Novartis: Consultancy, Honoraria; Ariad : Research Funding.

Recombinant, two-domain MHC Class II proteins (α1β1MHCII) that lack sequence from the membrane proximal α2 and β2 domains of MHC Class II adhere preferentially to B cells in vivo and ex vivo; whereas, full length (four-domain) constructs do not bind. Because α1β1MHCII molecules that contain only native MHC Class II sequence have preferential affinity for B cells, we hypothesize that a natural, cell-free form of MHC Class II (similar to recombinant α1β1MHCII) exists and has affinity for a receptor expressed by B cells. Evidence that endogenous, natural MHC Class II molecules transfer to B cells and that α1β1MHCII has inter- and intra-species affinity for B cells suggest that this is a natural and evolutionarily conserved property. And evidence that α1β1MHCII can stimulate CD4+ T cells suggests that there is an immune function for partial, cell-free MHC Class II. Transfer of immunoreactive, cell-free α1β1MHCII/Ag complexes to B cells suggests a here-to-fore unrecognized pathway for T cell activation.

SummaryStudying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.

Background Novel-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. Methods Primary 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. Results ALL 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. Conclusion These 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 material The online version of this article (doi:10.1186/s13045-015-0132-6) contains supplementary material, which is available to authorized users.