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The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

The CSF3R T618I Mutation Found In Chronic Neutrophilic Leukemia Removes An O-Linked Glycosylation Site and Increases Receptor Dimerization

CSF3R T618I Mouse Bone Marrow Transplant Model Of Neutrophilic Leukemia

BackgroundWe have recently identified mutations in CSF3R in the majority of patients with chronic neutrophilic leukemia (CNL). The most common mutation identified thus far is the T618I membrane proximal mutation, which confers rapid transformation capacity and ligand-independence to the receptor. We also observed less common mutations that result in truncation of the cytoplasmic domain of CSF3R. These truncation mutations, which have been studied in the context of severe congenital neutropenia progression to acute myeloid leukemia, lack the capacity for inducing disease in transgenic mouse models but enhance the progression to leukemia in the presence of a second genetic driver. The CSF3R T618I mutation transforms cells at a substantially faster rate than the truncation mutation, but the capacity of this mutation to induce a hematologic disorder in mice is not yet known. We created a CSF3R T618I mouse bone marrow transplant model, which resulted in a lethal myeloproliferative disorder characterized by high levels of granulocytes. MethodsMice were transplanted with donor bone marrow infected with a murine retrovirus expressing either wild type CSF3R or the CSF3R T618I mutation. White blood cell counts were measured 1-2 times per week for 90 days. The percentages of monocytes, granulocytes, T-cells and B-cells were analyzed by flow cytometery. The levels of pSTAT3 were measured by phospho-flow. Hematoxylin and eosin staining was performed on fixed and sectioned spleens, livers and bone marrow. ResultsMice transplanted with CSF3R T618I showed an initial transient granulocytic leukocytosis that normalized by 33 days post transplant. Subsequently, at day 47 the CSF3R T618I mice exhibited a dramatic and persistent rise in white blood cell counts. The elevated white blood cells were primarily mature granulocytes. By day 90, all of the mice transplanted with CSF3R T618I had died, while the mice transplanted with cells expressing wild type CSF3R did not exhibit morbidity or mortality. The granulocytes from CSF3R T618I mice also exhibited elevated levels of pSTAT3—a marker of JAK signaling downstream of CSF3R—relative to CSF3R wild type granulocytes. Histology revealed that both CSF3R wild type and T618I mice had hypercellular bone marrow, however the hypercellularity was more extreme in the CSF3R T618I mice. Mice harboring the CSF3R T618I and to a lesser extent wild type CSF3R exhibited infiltration of the spleen and liver by mature granulocytic cells and disrupted tissue architecture. ConclusionThe CSF3R T618I mouse bone marrow transplant provides a tractable model of Neutrophilic Leukemia. In contrast to CSF3R truncation mutations, which do not cause leukemia in isolation, the T618I mutation causes a fatal expansion of granulocytes. This model provides a useful tool for studying the biology of granulocytes and myeloproliferative neoplasms. Disclosures:Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Fleischman:Incyte: Speakers Bureau. Druker:Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other. Tyner:Incyte Corporation: Research Funding.

OP449, a Novel SET Antagonist, Is Cytotoxic To Leukemia Cells and Enhances Efficacy Of Tyrosine Kinase Inhibitors In Drug-Resistant Myeloid Leukemias

BackgroundThe SET oncoprotein, an inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia cells, preventing PP2A from performing its regulatory role in deactivating signaling proteins by dephosphorylation. Restoration of PP2A activity in both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells to normal levels through shRNA-mediated knockdown of SET results in reduced leukemogenesis. Given the central role of PP2A and SET in regulating various kinase-dependent and -independent downstream signaling pathways, we evaluated the efficacy of SET antagonism in CML and AML cell lines as well as primary patient cells using OP449, a novel, specific, cell-penetrating SET antagonist. ResultsTreatment of human and murine CML cells with OP449 resulted in dose-dependent increase in PP2A activity and selective inhibition of cell growth (IC50: 0.60 to 1.11 μM), while parental Ba/F3 cells exhibited no measurable cytotoxicity. OP449-mediated decrease in the viability of leukemia cells was significantly rescued by co-treatment with okadaic acid, a PP2A inhibitor, confirming efficacy is mediated through PP2A activation. OP449 was also 3 to 8-fold more potent than FTY720 (a known activator of PP2A) and induced dephosphorylation/degradation of BCR-ABL1, AKT, and STAT5. Importantly, OP449 demonstrated activity against the ABL1 tyrosine kinase inhibitor-resistant BCR-ABL1T315I mutant and the BCR-ABL1E255V/T315I compound mutant (IC50: 1.62 and 1.97 μM, respectively). Consistent with cell line findings, OP449 also inhibited growth of primary cells from CML blastic phase patients harboring either wildtype BCR-ABL1 or BCR-ABL1T315I while normal CD34+ cells exhibited minimal effect. Further, treatment of CML cell lines and primary CD34+ CML cells with OP449 in combination with the ABL1 tyrosine kinase inhibitors showed significantly increased cytotoxicity as compared to each compound alone. For example, treatment of primary CD34+ CML cells with 2.5 μM OP449 or 200 nM nilotinib alone each resulted in a 50% reduction in colony formation, while combination of OP449 and nilotinib at these concentrations reduced colony formation by approximately 87%, suggesting synergistic reduction of clonogenicity (combination index: 0.195). Similar to our findings in CML cells, OP449 increased PP2A activity and suppressed growth in a dose-dependent manner in AML cell lines and primary patient samples harboring various different genetic lesions including FLT3-ITD, CSF1R overexpression, NRASQ61L, and JAK3A572V. Additionally, synergistic inhibition of these cells was observed when OP449 was combined with relevant tyrosine kinase inhibitors and chemotherapy. For example, treatment of MOLM-14 cells (FLT3-ITD) with 2.5 μM OP449 or 1 nM AC220 alone reduced cell viability by 58% and 75%, respectively; combined treatment reduced cell growth 96% (combination index: 0.723). Similarly, treatment of HL-60 cells (NRASQ61L) with 1 μM OP449 or 250 nM cytarabine alone reduced cell viability by 40% and 60%, respectively, whereas combined treatment led to a 94% reduction in viability (combination index: 0.630). Mechanistically, AML patient samples showed significantly increased SET expression compared to normal CD34+ cells, and treatment of AML cells with OP449 reduced phosphorylation of downstream ERK, STAT5, AKT and S6 ribosomal protein signaling. Finally, to evaluate OP449 antitumor efficacy in vivo, we tested OP449 (5 mg/kg intraperitoneally every 3 days) in xenograft mice bearing human HL-60 cell derived tumors. OP449 significantly inhibited tumor growth measured over time and resulted in a >2-fold reduction in tumor burden at the end of the experiment compared to vehicle-treated controls (Day 18: 1.14±0.06 g vs. 0.45±0.08 g, respectively; p<0.001). These results demonstrate the in vivo efficacy of OP449 in a murine leukemia model. ConclusionsSET antagonism is selectively cytotoxic to CML and AML cells harboring various genetic lesions and drug-resistant mutations. Our results demonstrate that combined targeting of SET and tyrosine kinases provides more efficient and selective inhibition of leukemia cell growth for a broad range of oncogenic lesions as compared to normal cells. Taken together, our findings suggest a novel therapeutic paradigm of SET antagonism in combination with tyrosine kinase inhibitors for the treatment of CML and AML patients with drug resistance. Disclosures:Agarwal:Oncotide Pharmaceuticals: Research Funding. Tyner:Incyte Corporation: Research Funding. Vitek:Oncotide Pharmaceuticals: Employment. Christensen:Oncotide Pharmaceuticals: Employment. Druker:Ambit Biosciences: Consultancy, PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb/Novartis: Currently PI or co-I on Novartis & Bristol-Myers Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab Other; Oncotide Pharmaceuticals: Research Funding, Subaward from NIH STTR, Subaward from NIH STTR Other.

FGF2 Mediates Resistance In CML Patients In The Absence Of Kinase Domain Mutations, and Resistance Is Overcome By Ponatinib

BackgroundDevelopment of resistance to kinase inhibitors remains a challenge in chronic myeloid leukemia (CML). Kinase domain mutations are a common mechanism of resistance, yet the mechanism of resistance in the absence of mutations remains less clear. Recent evidence suggests that the bone marrow microenvironment provides a sanctuary for leukemia cells, and may be involved in mediating resistance to imatinib – particularly in the absence of BCR-ABL kinase domain mutations. We tested selected cytokines, growth factors, and extracellular matrix proteins expressed by cells in the bone marrow microenvironment for their ability to protect CML cells from imatinib. ResultsWe found that fibroblast growth factor 2 (FGF2) was the most protective protein for the K562 CML cell line when exposed to imatinib. FGF2 was not only capable of promoting growth in short-term culture, but uniquely able to promote long-term resistance in vitro (p<0.0001 by 2-way ANOVA analysis). To analyze the mechanism of resistance, we used siRNA to target the FGF receptors 1-4 and found that only siRNA targeting FGFR3 was able to abrogate the protective effect of FGF2. Phospho-chip and Western blot analysis revealed that FGF2 binds FGFR3, which then signals the downstream kinases Ras, c-RAF, MEK1, and ERK1/2 to promote survival in the presence of imatinib. Inhibition of FGFR3 with the specific FGFR inhibitor PD173074 led to dephosphorylation of this signaling cascade, and restored sensitivity to imatinib of FGF2-mediated resistant K562 cells. Resistance could also be overcome with ponatinib, a multi-kinase inhibitor that targets both BCR-ABL and FGFR, whereas imatinib, nilotinib and dasatinib were all ineffective against FGF2-mediated resistant K562 cells.Although ponatinib was rationally designed to circumvent the BCR-ABL T315I gatekeeper mutation, it was also able to achieve major cytogenetic responses in 62% of patients without detectable kinase domain mutations in the recent PACE trial. We theorized that increased FGF2 may drive resistance in the subset of patients without kinase domain mutations who respond to ponatinib, similar to our in vitro findings. To evaluate this possibility, we identified patients without kinase domain mutations who were responsive to ponatinib and quantified bone marrow FGF2 by immunohistochemistry. In comparison to ponatinib-responsive patients with kinase domain mutations, patients without kinase domain mutations had increased FGF2 in their bone marrow (50.5% versus 36.6%, p=0.033). Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGFR inhibition (-15.9% versus 0.8%, when compared to the change in FGF2 of patients with kinase domain mutations, p=0.012). Qualitatively, FGF2 was predominantly localized in supportive stromal cells (consistent with previous reports), supporting a paracrine mechanism of resistance. Furthermore, we also evaluated a single patient without kinase domain mutations who was resistant to ponatinib. In this patient's marrow, there was no elevation in FGF2 or change in FGF2 with ponatinib treatment. Taken together, inhibition of FGFR appears to be critical for the clinical activity of ponatinib in patients without kinase domain mutations. ConclusionsIn summary, our data supports a model of resistance in which FGF2 production by the marrow stromal cells promotes resistance to multiple ABL kinase inhibitors without the need for mutation of the ABL kinase domain. Resistance occurs via FGF2 ligand-induced activation of the FGFR3/Ras/MAPK pathway, and can be overcome by concomitant inhibition of ABL and FGFR. In combination with recent clinical data with ponatinib, our data suggest that FGF2-mediated resistance is a major mechanism of resistance in CML patients without kinase domain mutations. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance, particularly in other kinase-driven malignancies that routinely develop resistance to kinase inhibitors. Disclosures:Tyner:InCyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, & ARIAD: Novartis, BMS & ARIAD clin trial funding. OHSU holds contracts; no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict reviewed and managed by OHSU. Other.

Philadelphia Chromosome (Ph)-Positive Leukemia Patients Who Fail ABL1 Tyrosine Kinase Inhibitors Without BCR-ABL1 Kinase Domain Point Mutations Demonstrate Sensitivity To PI3-K/AKT Inhibitors

Despite the impressive success of imatinib in chronic myeloid leukemia (CML), resistance to therapy remains an issue for approximately 15-20% of newly diagnosed chronic phase patients by 5 years and transient responses are the rule for patients with blast crisis CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The most frequent and well-characterized mechanism of resistance to imatinib is acquisition of point mutations in the BCR-ABL1 kinase domain which compromise drug binding. This form of resistance is largely well-handled by the newer, more potent ABL1 inhibitors nilotinib, dasatinib, and ponatinib. However, in the remainder of patients with resistance sustained inhibition of BCR-ABL1 kinase activity is necessary but no longer sufficient to inhibit cell growth, implicating activation of additional, thus far, poorly understood BCR-ABL1-independent mechanisms of growth and survival. In an effort to identify and validate novel pathways important in BCR-ABL-independent resistance to ABL1 kinase inhibitors, we screened a cohort of 30 patients with CML or Ph+ ALL exhibiting clinical resistance to at least one ABL1 kinase inhibitor without evidence of a drug-resistant BCR-ABL1 kinase domain mutation. Following informed consent, fresh primary mononuclear cells were isolated from each sample by Ficoll gradient centrifugation, plated ex vivo in the presence of a panel of clinical and pre-clinical small-molecule kinase inhibitors, and assessed for effects on viability after 3 days. Ex vivo resistance to ABL1 kinase inhibitors largely tracked with clinical resistance profiles, and considerable variation in sensitivity profiles for other inhibitors was observed. Among these data, we found a particularly interesting subset of patients whose cells demonstrated ex vivo sensitivity to one or more PI3-K/AKT inhibitors (n=6/30; 20%) including PI-103, BEZ235, CAL-101, and MK-2206. These findings are consistent with previous studies demonstrating PI3-K activation in select ABL1 kinase inhibitor-resistant CML cell lines (Quentmeier et al., J Hem Onc 2011) and in response to imatinib in pre-overt resistance (Burchert et al., Leukemia 2005). Among the effective PI3-K/AKT inhibitors, the most prominent were the dual class IA PI3-K/mTOR inhibitor PI-103 (IC50 range:38-110 nM) and BEZ235 (IC50 range: 20-210 nM). To further interrogate the mechanism(s) behind dependence on this pathway, samples of interest were analyzed by deep sequencing using a custom capture library encompassing ∼2000 kinases, phosphatases, and adaptor proteins. However, in contrast to previous reports of activating PIK3CA mutations in imatinib-resistant KCL-22 cells, we found PIK3CA to be wild-type in all samples. Despite a common sensitivity to PI3-K/AKT pathway inhibition, we found sequence variants were somewhat heterogeneous and direct kinase targets in this pathway were wild-type, suggesting other causative lesions which activate this pathway. Variants were prioritized based on known associations to this pathway, generated and evaluated in vitro for Ba/F3 cell transformation capacity, and validated for pathway activation and kinase inhibitor sensitivity. These results will be presented. Taken together, our findings suggest that a subset of patients with Ph+ leukemia who become refractory to ABL1 kinase inhibitors without a BCR-ABL1 kinase domain mutation demonstrate acquired dependence on the PI3-K/AKT axis, warranting further investigation of inhibitors of this pathway alone and in combination with ABL1 inhibitors as a molecularly targeted therapeutic strategy in patients. Disclosures:Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Deininger:BMS: Consultancy, Research Funding; ARIAD: advisory board, advisory board Other, Consultancy; Novartis: advisory board, advisory board Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead: Research Funding. Tyner:Incyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, ARIAD & Incyte: Clin trial funding. OHSU holds contracts; Druker receives no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict has been reviewed and managed by OHSU. Other.

Long-Term Anti-Leukemic Activity of Ponatinib in Patients with Philadelphia Chromosome-Positive Leukemia: Updated Results from an Ongoing Phase 1 Study

Long-Term Anti-Leukemic Activity of Ponatinib in Patients with Philadelphia Chromosome-Positive Leukemia: Updated Results from an Ongoing Phase 1 Study

Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML

Inhibition of Autophagy in Combination with Ponatinib or Dual PI3K/mTOR Inhibition to Improve Treatment Response for Both Bcr-Abl Dependent and Independent Mechanisms of TKI-Resistance in CML

Integrative Analysis of Ikaros-Dependent Changes of Transcriptional Regulation and Tyrosine Phosphorylation Events in Ph+ ALL

AbstractAbstract 528 Background:Tyrosine kinase-driven B-cell lineage acute lymphoblastic leukemia such as Ph+ ALL, is associated with particularly poor clinical outcome and defined by deletions of the IKZF1 gene encoding the Ikaros transcription factor in >80% of the cases. While the role of Ikaros in lymphoid differentiation has been extensively studied, it remains elusive how Ikaros functions as tumor suppressor in Ph+ALL. Here we demonstrate that Ikaros orchestrates a set of gene expression changes that affects leukemia cell proliferation and survival at multiple levels. Results:A comprehensive gene expression analysis revealed that reconstitution of Ikaros function in patient-derived Ph+ ALL cells mimics gene expression changes induced by Pax5 (a B cell specific transcription factor), μ-chain and BLNK (pre-B cell receptor components), and treatment with Imatinib (a BCR-ABL1 kinase inhibitor), while it reverses Myc- and Stat5a/b-dependent survival signals. In combining seven experimental perturbations (reconstitution of Ikzf1, Pax5, μ-chain, Blnk, treatment with Imatinib or deletion of Stat5a/bfl/fl and Mycfl/fl), we identified a commonly regulated gene set, including known tumor suppressors SPIB, BTG1, and BTG2 and highly enriched for Myc-related gene signatures. These findings suggest that reconstitution of PAX5 and IKZF1 transcription factors converges with pre-B cell receptor-mediated transcriptional changes. We next connected our analysis at the transcriptional level with Ikaros-dependent changes at the level of tyrosine phosphorylation events: The BCR-ABL1 kinase in Ph+ ALL drives proliferation and survival through phosphorylation of Stat5, AKT, ERK, SRC and multiple other signaling intermediates. A global mass spectrometry analysis of phosphotyrosine proteins in patient-derived Ph+ ALL cells showed that reconstitution of Ikaros leads to global loss of phospho-tyrosine, in a comparable manner as treatment with TKI. The vast majority of Ikaros-mediated changes resulted in loss of tyrosine phosphorylation such as the SRC tyrosine kinase LCK (Y504 and Y192), the proximal BCR-ABL1 adapter molecule NEDD9 (Y166, Y240, Y344 and Y578) and ABL1 itself (Y257). For a small number of molecules, Ikaros-reconstitution resulted in increased tyrosine phosphorylation including the phosphatases Shp2 (Ptpn11Y584) and Ship2 (Inppl1Y1161). A focus phospho-tyrosine analysis of individual signaling molecules analysis revealed that Stat5, SRC, AKT and ERK are all affected by Ikaros-induced de-phosphorylation. These results suggest that that Ikaros induces dephosphorylation of multiple signaling intermediates in BCR-ABL1 signaling. To identify Ikaros-mediated gene expression changes that were directly induced by Ikaros promoter binding, we matched Ikaros-dependent gene expression changes with Ikaros ChIP-seq data. This analysis confirmed enriched binding of Ikaros on Btg1, Myc and Smyd2 promoters. Among Ikaros target genes, we identified a set of genes that predicts clinical outcome for adult and pediatric patients with ALL. According to this predictor, IkarosUP (FBXO33, HBP1, HIVEP1, DTX1, KIF13B, RHOQ) and IkarosDOWN (PFKP, ASNS, ETV6, PSTPIP2, SMYD2, CDKL5), patients with B-ALL were segregated into two groups with significantly different outcomes, with a 5-year RFS of 76% vs 29% (COG P9906 trial, n=110; p=0.006) and a 5-year OS of 39% vs 0% (ECOG E2993 trial, n=25; p=0.007). Further biochemistry and mouse experiments showed that both the ability of Ikaros to dephosphorylate Stat5 and to function as tumor suppressor requires cooperation with the pre-B cell receptor linker molecule BLNK but not the proximal effector kinase Syk. Conclusions:Importantly, Ikaros binds to and transcriptionally activates CDKN2A (Arf) and TP53 loci and thereby opposes the function of BCL6, which acts as transcriptional repressor at these loci. Comparing the gene expression microarray and ChIP-seq or ChIP-on-Chip data of BCL6 and Ikaros, we identified a set of target genes that is regulated by both BCL6 and Ikaros and found that for the vast majority of Ikaros/BCL6 target genes, Ikaros-reconstitution recapitulated a BCL6−/− situation, i.e. genes that are induced by Ikaros are repressed by BCL6. These findings are of potential clinical relevance since pharmacological inhibition of BCL6 restores sensitivity of IKZF1-mutant Ph+ ALL cells to conventional tyrosine kinase inhibitors. Disclosures:Druker:ARIAD: OHSU receives clinical trial funding. Dr. Druker is currently principal investigator or co-investigator on Novartis, Bristol-Myers Squibb, and ARIAD clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and da Other; Bristol-Myers Squibb: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; Novartis: OHSU receives clinical trial funding, OHSU receives clinical trial funding Other; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man, Scientific Founder; OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. This potential individual and institutional conflict of interest has been reviewed and man Other.

Combining Dose-Response Slope with in Vitro Sensitivity to ABL Tyrosine Kinase Inhibitors As an Improved Correlative Metric for Predicting Clinical Response in Chronic Myeloid Leukemia

AbstractAbstract 1670BCR-ABL mutations result in clinical resistance to ABL tyrosine kinase inhibitors (TKIs) in CML. Although in vitro IC50 values for specific mutations have been suggested to guide TKI choice in the clinic, quantitative relationship between IC50 and clinical response has never been demonstrated. Notably, IC50 value constitutes only one point on the dose-response curve for a given drug. Most dose-response curves can be described by Hill's equation (equation 1), which incorporates both IC50 and slope (m) parameters: [Display omitted] Here, fa is cell fraction affected by treatment and D is drug dose. We report estimation of the slope of in vitro dose-response curves for wild-type and kinase domain-mutant BCR-ABL against clinical ABL TKIs for CML and examine the value of this incorporated parameter for predicting clinical response. Dose response curves for imatinib, nilotinib and dasatinib were determined by methanethiosulfonate-based cell viability assay in Ba/F3 cells expressing wild-type BCR-ABL or each of 15 specific kinase domain point mutations (O’Hare T. et al Cancer Res 65:4500-5). The parameters m and IC50 were determined for each mutation and drug by fitting the log-transformed equation 1 to the respective dose-response curve. Excellent goodness of fit (r2 range 0.94–0.99) was observed for all drug-mutation pairings, confirming the adequacy of Hill’s equation to describe the effect of ABL TKIs on cellular viability in vitro. Inhibitory potential at peak concentration (IPP) was subsequently calculated as: [Display omitted] Here, fu is cell fraction unaffected by treatment, and D is mean peak concentration (Cmax) reported in plasma (Laneuville P. et al J Clin Oncol 28:e169-71). IPP and IC50 values for each Ba/F3 BCR-ABL mutant were compared with previously reported CCyR rates for nilotinib (Kantarjian H. et al Blood 110:3540-6) and dasatinib (Muller M.C. et al Blood 114:4944-53). Consistent with the particularly negative effect of P-loop mutations on drug binding and clinical outcome with imatinib, we found that 4 of 7 these mutations tested (G250E, Y253H, E255K, E255V) showed lower dose-response slope relative to wild-type BCR-ABL in addition to high IC50, while all other mutations showed variably increased slopes. Furthermore, the range of IPPs of imatinib for these mutations was lower than (and not overlapping with) all other mutations (0.084–1.66 vs 2.93–5.59; p=6*10−6). Slope variability provided particular additional interpretive value in cases where in vitro IC50 and clinical response are disparate. For example, G250E and V379I mutants feature comparable cellular IC50 values for imatinib (1184 and 1140 nM, respectively), but only G250E harbors worse clinical prognosis, arguably due to a lower dose-response slope of G250E, as also reflected in a lower IPP value compared to V379I (1.66 vs 3.04). Similarly, the M244V mutant does not confer substantial clinical resistance to imatinib despite increased IC50, possibly due to an exceptionally high slope value (m=5 vs. m=1.87 for wild-type BCR-ABL) reflecting a very steep dose-response curve which may render patients with this mutation particularly vulnerable to consequences of unfavorable imatinib pharmacokinetic profile or reduced compliance. Lastly, we examined whether higher IPP values were predictive of better clinical response to nilotinib and dasatinib in second-line clinical setting. Dasatinib-treated patients with mutations resulting in high IPP (IPP>7) had a significantly higher mean CCyR rate than those patients with lower IPPs (53% vs 31%; p=0.038). In contrast, this relationship was not evident when IC50 alone was used. In nilotinib-treated patients the difference in mean CCyR rate between patients with values above or below the median IPP or IC50 value approached but did not reach statistical significance (p=0.055 for both cases), potentially due to the lower number of patients for which response data by mutation has been reported (7 vs 11 mutations, 65 vs 295 patients with mutations, for nilotinib and dasatinib respectively). Taken together, our data suggest an integrated metric such as IPP may have both further relevance in conjunction with individual pharmacokinetic measurements and application to improved interpretation of mutationally-guided TKI treatment selection in CML and other malignancies. Disclosures:Vainstein:Neumedicines Inc: Employment. Druker:MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Dr Druker is a scientific founder, consultant, and stock holder in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD., OHSU and Dr. Druker have a financial interest in MolecularMD. Dr Druker is a scientific founder, consultant, and stock holder in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Other; Novartis, Bristol-Myers Squibb: Dr Druker's institution has contracts with these companies 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. Other.

Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Abstract▪2466▪This icon denotes a clinically relevant abstract BACKGROUND:Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS:To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS:The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION:The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures:Druker:MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; 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.; Cylene: Consultancy.

Resistance Profiling of BCR-ABL Compound Mutations Linked to Tyrosine Kinase Inhibitor Therapy Failure in Chronic Myeloid Leukemia

Abstract 1416Among patients with chronic myeloid leukemia (CML) who develop resistance to ABL tyrosine kinase inhibitor (TKI) therapy, the most common explanation is acquisition of point mutations within the BCR-ABL kinase domain that interfere with drug binding. The second-generation ABL TKIs nilotinib and dasatinib have proven effective against most imatinib-resistant mutants, with the critical exception of BCR-ABLT315I. Furthermore, sequential treatment with ABL TKIs can select for BCR-ABL compound mutations (two or more point mutations within the same BCR-ABL molecule) that are resistant to multiple inhibitors. With third-generation ABLT315I TKIs such as ponatinib (AP24534) and DCC-2036 currently in clinical development, containment of single point mutation-based resistance appears tenable. However, the ability of available inhibitors to address resistance due to the growing number of reported BCR-ABL compound mutations has not been investigated. Here, we generated stable Ba/F3 cell lines expressing a panel of clinically-observed BCR-ABL compound mutants (including two novel mutants which we identified from CML clinical resistance specimens, BCR-ABLG250E/V299L and BCR-ABLT315I/H396R) as well as cells expressing each constituent mutation. Cells were plated in graded concentrations of imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036, incubated for 72 hours, and cellular proliferation was analyzed by standard methanethiosulfonate-based assay. We found that, consistent with our previous cell-based mutagenesis screens for resistance (Blood 2006, 108: 2332–8; Cancer Cell 2009, 16: 401–12; Cancer Research 2011, 71: 3189–95), all five tested inhibitors demonstrated unique but partially overlapping resistance profiles relative to the panel of compound mutants tested. As expected, all mutants harboring a T315I component were insensitive to imatinib, nilotinib, and dasatinib, whereas ponatinib and DCC-2036 showed varying levels of efficacy against such mutants. The most resistant, clinically reported compound mutants were BCR-ABLG250E/T315I, BCR-ABLE255K/T315I, and BCR-ABLE255V/T315I, all of which conferred markedly increased resistance to ponatinib and DCC-2036 relative to cells expressing each constituent mutation alone (Table 1). For example, the most resistant compound mutant, BCR-ABLE255V/T315I, conferred high-level resistance to ponatinib and DCC-2036 (IC50: 425 and 1272 nM, respectively), while cells expressing either BCR-ABLE255V or BCR-ABLT315I were inhibited at clinically relevant concentrations of ponatinib (IC50: 33 and 18 nM, respectively) or DCC-2036 (IC50: 659 and 149 nM, respectively). Additional structural, biochemical, and signaling pathway analyses addressing the striking differences in sensitivity of BCR-ABL compound mutants versus their constituent mutations are being pursued and will be presented. Overall, our findings demonstrate that BCR-ABL compound mutations confer varying degrees of resistance to currently available ABL TKIs, necessitating rational treatment selection to optimize outcomes. Patients harboring highly multi-drug-resistant compound mutants such as BCR-ABLE255V/T315I may have limited therapeutic options available, warranting investigation into combination therapies involving ABLT315I TKIs. Furthermore, studies of compound mutation-based resistance mechanisms in CML advance the possibility of maximum disease control in a greater proportion of patients and have important implications for anticipating resistance and designing treatment strategies in similar, more rapid clinical resistance scenarios such as those encountered with EGFR or ALK mutations in non-small-cell lung cancer.Table 1Cell proliferation IC 50s for clinically-observed BCR-ABL compound mutants.Cell lineimatinib (nM)nilotinib (nM)dasatinib (nM)Ponatinib (nM)DCC-2036 (nM)Parental>5120>5120>768>768>5120Native BCR-ABL270121.22.337BCR-ABLG250E26391702.87.7180BCR-ABLG250E/T315I>5120>5120>76849627BCR-ABLE255K>51203268.923410BCR-ABLE255K/T315I>5120>5120>768106585BCR-ABLE255V>512011771333659BCR-ABLE255V/T315I>5120>5120>7684251272BCR-ABLT315I>5120>5120>76818149 Disclosures:Druker: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.; 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.; 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.

Mutationally Activated TYK2 From T-ALL Specimens Exhibits Tranformative Capacity in Cell Lines and Primary Cell Models and T-Lineage Expansion in Mice

Abstract 74 Background:First-line therapy for T-cell acute lymphoblastic leukemia (T-ALL) fails in 25% of children and more than 50% of adults despite improvements in clinical outcomes due to intensification of therapy. Development of new and improved therapeutic regimens will require 1) new gene targets relevant to the pathogenesis of T-ALL, 2) targeted therapeutic agents with activity against those gene targets, and 3) effective models for pre-clinical development of these gene targets and therapies. Current in vivo models of T-ALL recapitulate derangements in the Notch signaling pathway but modeling alternative pathways of T-ALL leukemogenesis are lacking. We report that tyrosine kinase 2 (TYK2) is vital for the maintenance of T-ALL growth and viability, and we have developed a mouse model based on an activating mutation of TYK2 found in a T-ALL specimen. This mouse model will be useful for further exploration of the biological role of TYK2 in T-ALL pathogenesis as well as pre-clinical evaluation of TYK2 inhibitors as components of T-ALL therapy regimens. Methods: Sequence analysis of TYK2 in T-ALL cell lines and patient samples revealed a diversity of TYK2 point mutations. These mutations were located just outside of or within the FERM domain (V15A, G36D, G36R, S47N, R425H), the pseudokinase domain (V731I), and the kinase domain (E957D, and R1027H). Identified point mutant were cloned and introduced into Ba/F3 cells for assessment of transformative capacity. The point mutant E957D was ectopically expressed via retroviral transduction in primary murine bone marrow hematopoietic progenitor cells and co-cultured on OP9-DL1 stromal cell layers to assay the effect of this mutation on in vitro T-lineage development and expansion. A murine transduction/transplantation model was developed to investigate the effect of TYK2 E957D in vivo. Results: Introduction of the point mutants into Ba/F3 cells revealed that 7/8 (all except R425H) transformed cells to factor-independent growth. Transformed cells displayed constitutive phosphorylation of TYK2, STAT1, STAT3, and ERK1/2. Treatment of transformed cells with JAK Inhibitor I revealed extreme sensitivity of cells to this TYK2 kinase inhibitor as well as reduction of TYK2 AND STAT1 phosphorylation. Ectopic expression of TYK2 E957D enhanced in vitro T cell development and subsequent expansion of mature T cells on OP9-DL1 stromal cell layers as compared to TYK2 WT or empty vector. Fifty percent of mice transplanted with bone marrow ectopically expressing TYK2 E957D died at 7 months post-transplant with evidence of hematologic malignancy characterized by massive expansion of the thymus and infiltration of the bone marrow with a monomorphic population of cells. In comparison, none of the mice transplanted with wild-type TYK2 were adversely affected. Conclusions: We demonstrate that mutations in TYK2 in T-ALL can confer constitutive activity to the TYK2 signaling pathway, which can play a functional role in the pathogenesis of T-ALL. Ectopic expression of TYK2 E957D leads to enhanced expansion of T cells and mice expressing this point mutant in the hematopoietic compartment die from hematologic malignancy with grossly enlarged thymuses after a long period of latency. This model could complement the arsenal of currently available T-ALL models and may represent and the latency of disease may allow a search for cooperating elements leading to transformation. Small-molecule kinase inhibitors with activity against TYK2 reduce the growth of these TYK2-dependent cells. Hence, we have defined a new, oncogenic signaling pathway in T-ALL cells that can be readily modulated by kinase inhibitors. Cumulatively, these data offer new therapeutic options for T-ALL patients while simultaneously providing novel insights into the biology of T-cells and the TYK2-STAT1 signaling cascade. Disclosures:Druker:Novartis:; Bristol-Myers Squibb:; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Cylene: Consultancy.

Cryptic Intracellular Retention of ABL Tyrosine Kinase Inhibitors within CML Cells Mediates Apoptosis Commitment Following Acute Drug Exposure,

Abstract 3504The 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.

Subset Analysis of Response to Treatment of Chronic Phase CML in a Phase 1 Study of Ponatinib in Refractory Hematologic Malignancies

Abstract 602 Background:Ponatinib is a potent, oral, pan-BCR-ABL inhibitor active against the native enzyme and all tested resistant mutants, including the uniformly resistant T315I mutation. Initial findings of a phase 1 trial in patients (pts) with refractory hematologic malignancies have been reported. The effect of duration of treatment, prior treatment, and mutation status on response to treatment was examined in CML chronic phase (CP) pts who responded to ponatinib. Methods: An open-label, dose escalation, phase 1 trial of ponatinib in pts with hematologic malignancies is ongoing. The primary aim is to assess the safety; anti-leukemic activity is also being investigated. Pts resistant to prior treatments or who had no standard treatment available were enrolled to receive a single daily oral dose of ponatinib (2 mg to 60 mg). Subset analyses of factors impacting cytogenetic and molecular response endpoints (MCyR and MMR) were performed for pts with CP-CML. Data are presented through April 15, 2011. Results: In total, 81 pts (54% male) received ponatinib. Overall, 43 pts had CP with 34 ongoing at analysis. MCyR was observed as best response in 31/43 (72%), 27 (63%) CCyR. The median time to MCyR was 12 (3 to 104) wks. Response rates were assessed by duration of treatment (1 pt in CCyR at entry was excluded; 6 pts in PCyR had to achieve CCyR). At the 3 month assessment, 22/42 (52%) CP pts achieved MCyR; at 6 months, 24/42 (57%); at 12 months, 29/42 (69%) had MCyR. The impact of prior treatment on response and time to response was assessed. 42 pts (98%) had >2 prior TKIs and 28 (65%) ≥3 prior TKIs, including investigational agents. Of approved TKIs, all pts were previously treated with imatinib, 19 dasatinib or nilotinib after imatinib, and 21 both dasatinib and nilotinib after imatinib. MCyR rate decreased with number of prior TKIs (2 prior TKIs 13/14 [93%], ≥3 prior TKIs 17/28 [61%]) and number of approved TKIs (imatinib followed by dasatinib or nilotinib 17/19 [90%], or by both dasatinib and nilotinib 12/21 [57%]). Time to response was prolonged in pts more heavily treated with prior TKIs. Median time to MCyR increased with the number of prior TKIs and approved TKIs (2 TKIs 12 wks, ≥3 TKIs 32 wks). The effect of mutation status on response and time to response was also evaluated. At entry, 12 pts had the T315I mutation, 15 had other BCR-ABL kinase domain mutations, 12 had no mutations detected, 4 did not allow sequencing. MCyR response rate for CP pts with T315I was 11/12 (92%); for other mutations, 10/15 (67%); and no mutation, 7/12 (58%). Similarly, mutation status had an impact on time to response: median time to MCyR was 12 wks for those with T315I or other mutations and 32 wks in resistant pts with no mutation. All CP patients were evaluable for MMR. At analysis, MMR was 17/43 (40%). MMR rate was inversely related to number of prior TKIs (2 TKIs 10/14 [71%], ≥3 TKIs 6/28 [21%]), approved TKIs (imatinib followed by dasatinib or nilotinib 12/19 [63%], or by both dasatinib and nilotinib 4/21 [19%]), and was higher for T315I pts (7/12, 58%) and those with other mutations (7/15, 47%) compared with no mutation (2/12, 17%). Median time to MMR for CP pts was 97 wks; median time to MMR was shorter for pts who were less heavily treated (2 prior TKIs 24 wks) and those with T315I or other mutations (63 wks). Conclusion: In this subset analysis of the phase 1 data, ponatinib had substantial activity in all subgroups analyzed. Time on treatment, less prior therapy and kinase domain mutations were associated with higher response rates and early responses in CP pts. Cytogenetic responses improved over the first 12 months of treatment and were higher in less heavily treated pts. Disclosures:Cortes:Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; ARIAD: Research Funding. Shah:Ariad: Consultancy, Research Funding. Bixby:Novartis: Speakers Bureau; BMS: Speakers Bureau; GSK: Speakers Bureau. Mauro:ARIAD: Research Funding. Flinn:ARIAD: Research Funding. Hu:ARIAD: Employment. Clackson:ARIAD: Employment, Equity Ownership. Rivera:ARIAD: Employment, Equity Ownership. Turner:ARIAD: Employment, Equity Ownership. Haluska:ARIAD: Employment, Equity Ownership. Druker:MolecularMD: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t. 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. Talpaz:ARIAD: Research Funding.

A Novel SET Antagonist (OP449) Is Cytotoxic to CML Cells, Including the Highly-Resistant BCR-ABLT315I Mutant, and Demonstrates Enhanced Efficacy in Combination with ABL Tyrosine Kinase Inhibitors,

Abstract 3757 Background:The majority of patients with chronic myeloid leukemia (CML) achieve durable remissions with ABL tyrosine kinase inhibitor (TKI) therapy. However, a subset of CML patients develop resistance to ABL TKIs, either through BCR-ABL kinase-dependent (e.g. kinase domain point mutations) or BCR-ABL kinase-independent (e.g. overexpression of LYN) mechanisms. Recent studies have demonstrated that the presence of BCR-ABL in CML cells result in overexpression of the protein phosphatase 2A (PP2A) inhibitor SET, which in turn prevents PP2A from performing its regulatory role in deactivating signaling proteins through dephosphorylation. Further, loss of PP2A function accounts for increased and sustained BCR-ABL activity in CML blast crisis cells and activation of PP2A via treatment with Forskolin or FTY720 reduces clonogenicity of CML cells, regardless of their sensitivity to ABL TKIs. Therefore, given the central role of PP2A in regulating both BCR-ABL kinase-dependent and kinase-independent downstream signaling, pharmacological activation of PP2A may provide a novel approach to the development of CML therapeutics, particularly for patients with imatinib-resistant CML. Results:Here, we report a novel, specific, cell penetrating peptide known as OP449 that binds to SET and antagonizes SET’s inhibition of PP2A. Initial in vitro studies of OP449 in human (K562) and murine (32Dp210) CML cell lines revealed a robust (45%) increase in PP2A activity after treatment with 1μM OP449 when compared to vehicle treated cells. OP449 was more potent than the PP2A activator FTY720 (5μM; 20% increase in PP2A activity compared to vehicle treated control). In comparative cytotoxicity studies, treatment with OP449 consistently resulted in reduced proliferation and increased apoptosis compared to FTY720 in K562 (IC50: 2.5μM vs. 5μM, respectively) and LAMA (IC50: 1.25μM vs. 2.5μM, respectively) cells. Consistent with the role of OP449 as a PP2A activator, its growth inhibitory effects in CML cells were rescued by treatment with okadaic acid, a PP2A inhibitor, indicating that OP449 specifically activates PP2A. Furthermore, the cytotoxic effects of OP449 are more selective for BCR-ABL-positive cells (K562, LAMA, 32Dp210 IC50 range: 1.0 –2.5μM) relative to parental 32D cells (IC50: 5μM). Importantly, TKI-resistant mutant CML cells were also sensitive to OP449 at similar doses (32Dp210T315I cells IC50: 2.5μM). Similarly, in primary CML CD34+ cells, we noted CFU-GM colony formation was reduced by 75% and 98% with 2.5 μM and 5 μM OP449, respectively, while no toxicity was observed in normal human CD34+ cells at concentration of 5 μM. Intriguingly, the combination of OP449 with ABL TKIs (imatinib 1.0μM, nilotinib 50nM, or dasatinib 5nM) further reduced proliferation and increased apoptosis in K562 cells and in primary CD34+ CML cells combination of 2.5 μM OP449 with nilotinib (50nM) reduced colony formation by 98%, which was more than treatment with each drug alone. These data support the potential use of OP449 in combination with ABL TKIs.Mechanistically, the amount of SET protein remained unchanged with OP449 treatment, although levels of CIP2a, another PP2A inhibitor, were reduced significantly. OP449 also inhibited BCR-ABL-mediated downstream signaling, and induced dephosphorylation and degradation of BCR-ABL, STAT5, AKT, ERK, and p38/MAPK in CD34+ CML cells and K562 cells within 6 hr of treatment. Lastly, in vivo toxicity studies did not identify any toxicity of OP449 in non-leukemic mice up to 10mg/kg/day. In a 32Dp210 xenograft model, mice treated with OP449 survived slightly but significantly longer than vehicle-treated mice (p=0.05). In a murine CML bone marrow transplantation model, increasing doses of OP449 correlated with improved survival, reduced WBC count, and reduced GFP+ leukemic cells. Combination treatment of OP449 with nilotinib showed further reduced leukemia burden as compared to monotherapy. Conclusions:Taken together, these data demonstrate that OP449 is selectively cytotoxic to CML cells both in vitro and in vivo by antagonizing SET and increasing PP2A activity, thus restoring PP2A's tumor suppressor function. Furthermore, these data validate SET antagonism as a target for development of new CML therapeutics for treatment of CML patients with resistance to ABL TKIs either alone or in combination with ABL TKIs. Disclosures:Oddo:Oncotide: Employment, Research Funding. Vitek:Oncotide: Employment, Equity Ownership. Christensen:Oncotide: Employment, Equity Ownership. Druker:Bristol-Myers-Squibb: OHSU has clinical trial contracts with Novartis and Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead.; Novartis: OHSU has clinical trial contracts with Novartis and Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead.; MolecularMD: Equity Ownership, verbatim: OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Commi; Cylene: Consultancy; Ariad pharmaceuticals: Consultancy.