Chronic Phase CML Research Articles

Relative increase in lymphocytes from as early as 1 month predicts improved response to dasatinib in chronic-phase chronic myelogenous leukemia

Lymphocytosis in response to dasatinib for chronic myelogenous leukemia (CML) may be associated with favorable response. However, it occurs at varying times and in a limited subset of patients. To identify early clinical markers for favorable responses applicable to all patients with or without lymphocytosis, we prospectively analyzed lymphocyte profiles of 50 Japanese CML patients treated with dasatinib after intolerance/resistance to imatinib. Although absolute lymphocyte counts did not differ significantly until 3 months between patients with complete molecular response (CMR) at 12 months and those without it, relative increases in lymphocyte compared with baselines differed significantly from 1 month. Patients with relative lymphocyte counts >150 % at 1 month or >200 % at 3 months had higher CMR rates at 12 months than others (57.9 vs. 23.3 %, P = 0.015, and 76.5 vs. 16.1 %, P < 0.0001, respectively). A relative increase in lymphocyte subset of CD57(+)CD14(-), CD8(+)T, or NK cells >200 % at 1 month was also significantly associated with a higher CMR rate. There were significant negative correlations between relative lymphocyte increases and BCR/ABL transcript levels. CD57(+)CD14(-) cells were a highly specific focus of proliferation. Relative increases in lymphocyte count and its subsets from 1 month are reliable early markers of favorable responses to dasatinib.

Bosutinib

To review clinical trials and main characteristics of bosutinib, a second-generation tyrosine kinase inhibitor (TKI) for treatment of chronic myelogenous leukemia (CML). Pertinent data were identified through a search of PubMed (January 1990-April 2013) using the primary search terms SKI-606, bosutinib, and CML. Additionally, preliminary reports published in abstract form by the American Society of Clinical Oncology and American Society of Hematology (January 1990-April 2013) were screened for inclusion. Clinical Phase 1, 2, and 3 studies reported in English evaluating the safety and efficacy of bosutinib in patients with CML were reviewed. Bosutinib is a TKI of the breakpoint cluster region/Abelson murine leukemia (BCR-ABL) gene approved by the Food and Drug Administration on September 4, 2012, for second-line treatment of chronic phase, accelerated phase, and blast phase CML. In the second-line setting, bosutinib is effective in some patients with CML resistant or intolerant to imatinib, dasatinib, and/or nilotinib, but it is not effective in patients whose disease expresses the T315I point mutation in BCR-ABL. Bosutinib also has been compared with imatinib, the standard first-line treatment, in 502 patients with newly diagnosed chronic phase CML in a Phase 3 trial. Complete cytogenetic response at 12 months, the primary efficacy end point, is similar between bosutinib and imatinib (p = 0.601); therefore, bosutinib is not indicated in the first-line setting. Common adverse events associated with bosutinib include diarrhea, nausea, and vomiting. Grade 3 and 4 adverse events reported in at least 5% of bosutinib-treated patients include elevated serum lipase and liver aminotransferases, anemia, thrombocytopenia, neutropenia, and diarrhea. Currently available clinical trials suggest that bosutinib is generally a safe and effective treatment option for patients with CML who have failed first-line TKIs and who do not express the T315I mutation; however, tolerability may be problematic for some patients.

Nilotinib 300 mg BID as frontline treatment of CML: Prospective analysis of the Xpert BCR-ABL Monitor system and significance of 3-month molecular response

Sixty patients with early chronic phase CML (ECPCML) received Nilotinib on a phase II study which included a comparison of the Xpert BCR-ABL Monitor™ PCR system with standardized (IS) BCR-ABL1 real-time quantitative PCR (RQ-PCR). 88% patients achieved MMR with 45% achieving MR4.5. At 3 months BCR-ABL1/ABL1 IS >1% and <10% was associated with a lower likelihood of subsequent MR4.5 compared to patients with lower levels (p = 0.018). No significant difference was observed between methodologies in identifying MMR. Nilotinib induces high molecular response rates in ECPCML and the Xpert BCR-ABL Monitor™ system merits further investigation in this setting.

Primitive CML cell expansion relies on abnormal levels of BMPs provided by the niche and on BMPRIb overexpression

AbstractLeukemic stem cells in chronic phase chronic myelogenous leukemia (CP-CML) are responsible for disease persistence and eventual drug resistance, most likely because they survive, expand, and are sustained through interactions with their microenvironment. Bone morphogenetic proteins 2 (BMP2) and 4 (BMP4) regulate the fate and proliferation of normal hematopoietic stem cells, as well as interactions with their niche. We show here that the intrinsic expression of members of the BMP response pathway are deregulated in CML cells with differences exhibited in mature (CD34−) and immature (CD34+) compartments. These changes are accompanied by altered functional responses of primitive leukemic cells to BMP2 and BMP4 and strong increases in soluble BMP2 and BMP4 in the CML bone marrow. Using primary cells and a cell line mimicking CP-CML, we found that myeloid progenitor expansion is driven by the exposure of immature cells overexpressing BMP receptor Ib to BMP2 and BMP4. In summary, we demonstrate that deregulation of intracellular BMP signaling in primary CP-CML samples corrupts and amplifies their response to exogenous BMP2 and BMP4, which are abnormally abundant within the tumor microenvironment. These results provide new insights with regard to leukemic stem cell biology and suggest possibilities for the development of novel therapeutic tools specifically targeting the CML niche.

The Poison Oligonucleotide F10 Is Efficiently Taken Up By, and Highly Effective Against, Acute Lymphoblastic Leukemia While Sparing Normal Hematopoietic Cells

The development of imatinib to target the BCR-ABL kinase in chronic phase (CP) CML has changed the natural history of the disease. This success generated much enthusiasm for the approach in the more genetically complex acute lymphoblastic leukemia (ALL). Unfortunately, the durable responses seen in CP CML are not reproduced in BCR-ABL positive ALL. Indeed, all previous attempts to target a single oncogenic pathway in acute leukemias have resulted in transient responses with frequent relapse. An alternative approach is to use agents that target “final common pathways”, i.e. processes that must be accomplished to produce additional leukemia cells regardless of driving mutations. In this paradigm, agents are not judged by differential expression of a target, but by the degree of differential uptake. One pathway to exploit is the known increased uptake of oligonucleotides by ALL cells.F10 is a poison deoxy-oligonucleotide that is a 10mer of the thymidylate synthase (TS) inhibitory 5-fluorouracil (5-FU) metabolite, 5-fluoro-2'-deoxyuridine-5'-O-monophosphate. We sought to determine the uptake and activity of F10 against preclinical ALL models.Using fluorescently labeled F10, we determined that uptake by the human ALL cell lines DG75 and SUP-B15 was rapid and had a profound temperature dependence consistent with an active process. Both cell lines demonstrated increased uptake compared to normal, lineage- depleted marrow cells from C57Bl/6 mice. Using a syngeneic, BCR-ABL-expressing murine ALL model, we confirmed rapid uptake of F10 in vivo. Furthermore, preliminary experiments suggested normal human HSCs have decreased uptake compared to primary patient samples. Consistent with this decreased uptake, F10 treatment did not alter the ability of human HSCs to engraft in immunodeficient mice.F10 treatment resulted in robust induction of apoptosis that could not be equaled by 100 fold more 5-FU. IC50 values for F10 against B6 ALL, Jurkat, DG75, Molt-4, CCRF-CEM, and SUP-B15 were in the picomolar to nanomolar range, with an average value of 2.15 nM (range 0.12 to 5.4 nM). For comparison we also tested 5-FU, doxorubicin, and cytarabine. In all cases, F10 was the most potent agent; over 1000 times more potent than 5-FU.In vivo, F10 treatment was associated with a significant increase in survival in a BCR-ABL driven, syngeneic ALL mouse model (p= 0.0001 by log rank test). Survival was dose-dependent, with median survival extended by 3, 9 or 15 days for 4, 6, or 9 doses, respectively. F10 also protected mice from leukemia-induced weight loss during and for several days after treatment. To confirm the in vivo activity of F10, we treated a separately derived syngeneic model expressing the T315I variant of BCR-ABL. As in the previous model, F10 significantly prolonged survival (p=0.0013). To further extend these results and confirm F10's activity against human ALL cells, we treated DG75, Jurkat, and SUP-B15 xenograft models. As in the murine models, F10 caused regression of disease with several animals cured and resulted in a significant survival benefit (p=0.0112).As the uptake and mechanism of action of F10 is unique we sought to determine the in vivo efficacy of F10 on ALL previously treated with cytarabine. When mice injected with previously treated ALL cells were treated with cytarabine a median survival benefit of only one day (11 vs 12 days) was observed. In contrast F10 treatment resulted in a median survival of 31 days, similar to the survival seen in previously untreated ALL.F10 exposure results in trapped topoisomerase I (Topo1) cleavage complexes in vitro. To determine if this occurs in vivo, we treated leukemic C57Bl/6 or nude mice harboring ALL xenografts with F10. After 24 hours, leukemia cells were harvested and assayed for trapped Topo1 complexes. F10 treatment resulted in detectable trapped Topo1 complexes in both the xenograft and syngeneic models.In summary, F10 exhibited remarkable activity against human and murine ALL cells in vitro and in vivo by inducing apoptosis and trapping Topo1 complexes. These data demonstrate agents that target “final common pathways” but with differential uptake can be safe and effective, even against genetically complex and aggressive leukemias. Disclosures:Pardee:Salzburg Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gmeiner:Salzburg Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Relative RATIO Of BCR-ABL1 Transcript Clearance Improves The Prognostic VALUE Of Single PCR Assessment At The 3 and 6 Month Time Points In Patients With Chronic Myeloid Leukemia In Chronic PHASE Treated In The Dasision Study

BackgroundA cut-off value of 10% BCR-ABL1IS has been retrospectively validated by several groups as predictive of favorable long-term outcomes. The NCCN guidelines establish that transcripts >10% represent failure to TKI therapy that warrants a change in therapy. It remains controversial whether the predictive value of combining PCR values at 3 and 6 mos improves that of the use of a single PCR value at 3 mos. We aimed to improve the accuracy of early time point PCR prognostication by using the fractional clearance of BCR-ABL1 transcripts between baseline and 3 mos and between 3 and 6 mos in pts with CML in chronic phase (CML-CP) treated in the DASISION study. MethodsPts with CML-CP were randomized to receive dasatinib (DAS) 100 mg QD (n=259) or imatinib (IM) 400 mg QD (n=260). Methods have been previously reported (NEJM 2010 362 2260). PFS rates were obtained from Kaplan-Meier estimates. Qualifying events for PFS included: increasing WBCs, loss of complete hematologic response or major cytogenetic response, transformation to AP/BP, or death. Outcomes of pts reaching similar PCR milestones were similar regardless of therapy and they were merged for global outcome analyses. ResultsWe first evaluated the prognostic value of combining PCR values at 3 and 6 mos in the DASISION study. Overall, 473 pts (234 IM, 239 DAS) remained progression-free at 3 mos and 464 (233 IM, 231 DAS) at 6 mos with PCR assessments available. We used as a cut-off transcript levels determined by ROC analysis that optimally predicted for PFS at 3 months (lower or higher than 10.04%) and 6 months (lower or higher than 2.34%).(3-year PFS for pts receiving IM or DAS was 87.0% vs 87.9%, respectively. A total of 103 (45.6%) pts on IM and 150 (65.8%) on DAS had low transcripts both at 3 and 6 mos (LO/LO) and their 3-year PFS was 98.0% vs 92.5%, respectively (94.7% for both cohorts combined). Seventy-seven (34.1%) IM treated pts and 33 (14.5%) DAS treated pts had high transcript levels (HI/HI) at 3 and 6 mos and their 3-year PFS was 72.0% (p<0.0001) for IM, 75.9% (p<0.127) for DAS (73.2%, p<0.0001 for combined dataset). Seventy-five pts (40 (17.7%) IM, 35 (15.3%) DAS) pts were classified as LO/HI and had PFS of 89.7% (IM), 88.1% (DAS) and 88.9% (merged dataset). Only 16 (6 IM, 10 DAS) pts were HI/LO but none experienced progression (similar to LO/LO pts). Thus, combining PCR values at 3 and 6 mos added little prognostic discrimination to the single 3-mo PCR assessment.We then examined the prognostic value of combining PCR values at 0 and 3 mos (i.e. rate of transcript elimination during the first 3 mos of therapy, Table 1, Figure 1). The relative ratio at 3 mos (RR3) was defined as: (PCR at 0 mos-PCR at 3 mos/PCR at 0 mos), while RR6 was: (PCR at 3 mos-PCR at 6 mos/PCR at 3 mos). ROC analysis established cut-off values of 64.5% at 3 mos and 77.5% at 6 mos. Of note, at 3 mos, BCR-ABL1<10.04% and RR3<64.5% identified 14 pts with 3-year PFS of only 79% (vs 90% for those with RR3 >64.5%) in spite of having achieved an optimal response by standard criteria (i.e. PCR<10%). Similarly, at 6 mos, BCR-ABL1<2.34% and RR6<77.5% identified a group of pts with PFS of 87% (vs 97% for those with RR6>77.5%).Table 1Prognostic impact of BCR-ABL1 relative ratio at 3 and 6 months on PFS.3 month PCRRR3 mo, No. pts (3-year PFS, %)Total>64.5%<64.5%<10.04%325 (90)14 (79)339 (89%)>10.04%38 (86)96 (67)134 (73%)Total363 (90)110 (69)4736 month PCRRR6 mo, No. pts (3-year PFS, %)Total>77.5%<77.5%<2.34%199 (97)129 (87)328 (93%)>2.34%18 (94)118 (70)136 (73%)Total217 (96)247 (79)464 [Display omitted] ConclusionThe combined use of cut-off PCR values at 3 and 6 mos do not significantly improve the prognostic value of a single 3 mo PCR assessment. However, the combination of these cut-offs with the RR of BCR-ABL1 transcript decrease between 0 and 3 mos and between 3 and 6 mos identifies pts with unfavorable outcomes among those with optimal PCR values at 3 and 6 mos, and pts with favorable outcomes among those with suboptimal PCR values at 3 and 6 mos. Validation of these data using an independent dataset of pts treated with imatinib at our institution is underway. Disclosures:Quintás-Cardama:Novartis, BMS, Ariad: Consultancy. Cortes:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding.

The BCL-2 Inhibitor ABT-199 Enhances Imatinib-Induced Cell Death In Chronic Phase CML Progenitors

The advent of tyrosine kinase inhibitors (TKI) that specifically target BCR-ABL has significantly prolonged the life of patients with CML. We and others have previously shown that the BH3 mimetic ABT-737 or its clinical equivalent, ABT-263, can significantly enhance TKI-induced cell death in both CML cell lines and primary cells (Ng et al. Nature Medicine, 18: 521-528, 2012). ABT-737 and ABT-263 are BH3 mimetics that have broad specificity against anti-apoptosis regulators such as BCL-2 and its related proteins (BCL-XL & BCL-W). However, in clinical trials employing ABT-263, a dose-limiting toxicity is thrombocytopenia. This is due to the inhibitory effect of ABT-263 on BCL-XL that is pivotal for platelet survival. As a result, a new BH3 mimetic, ABT-199, has been developed that binds with high affinity to BCL-2 but not BCL-XL, and thus does not harm platelets.Here, we evaluated the effectiveness of ABT-199, as a single agent or in combination with imatinib, in reducing the viability of CD34+ cells from the different phases of CML (chronic, accelerated and blast). We also assessed the cytotoxic effect of ABT-199 on normal cord blood CD34+ cells. The number of samples used in this study was: (1) chronic phase CML (CP, n=4), (2) accelerated phase CML (AP, n=2), (3) blast phase CML (BP, n=1), and (4) normal cord blood (NCB, n=3). We evaluated the viability of progenitor cells by colony formation assay. A broad concentration range of ABT-199 (in nM: 0, 1, 2, 5, 10, 20, 50, 100, 200, 500, 2000) was used in this study. The concentration of imatinib used was 2uM, which is in line with the plasma concentrations achievable in most patients with CML.Below are three tables that summarize the results obtained from this study.Table 1Treatment with 2uM imatinib only.CP (n=4)AP & BC (n=3)NCB (n=3)Average CFU reduction65%23%20%(Relative to DMSO control)Table 2Treatment with ABT-199 only: concentration required to achieve the following CFU reduction.Average CFU reductionCP (n=4)AP & BC (n=3)NCB (n=3)50%500nM2000nM10nM90%2000nMNot achieved within the concentration range used500nMTable 3Combined treatment with ABT-199 and 2uM imatinib (IM): concentration required to achieve the following CFU reduction.Average CFU reductionCP (n=4)AP & BC (n=3)NCB (n=3)50%Not achieved within the concentration range used2nM ABT-199 + 2uM IM10nM ABT-199 + 2uM IM90%10nM ABT-199 + 2uM IM200nM ABT-199 + 2uM IM100nM ABT-199 + 2uM IMIn summary, we found that: (1) Compared to CML, NCB progenitors were very sensitive to ABT-199, with a reduction of average cell viability to 50% at just 10nM (with or without imatinib); (2) When the dose-limiting cytotoxic effect of ABT-199 on NCB was taken into consideration, we were able to discover an ABT-199 concentration (10nM) that significantly enhanced imatinib-induced cell death of CP CML progenitors without causing NCB viability to fall below 50%; and (3) The inferior efficacy of the combination on AP/BC vs CP progenitors suggests that BCL2-independent mechanisms contribute to the survival of advanced phase CML progenitors.Finally, our in vitro data may also explain some of the adverse hematologic effects observed in clinical trials that employ ABT-199, including neutropenia and thrombocytopenia. Nevertheless, our data suggest that ABT-199, when used at a concentration that is not cytotoxic to normal progenitors and in combination with imatinib, can be beneficial in treating patients with chronic phase CML. Disclosures:Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Cytokine-STATs Signalings Upregulate C/EBPβ In BCR-ABL+ Leukemic Cells Independently From BCR-ABL/JAK-STAT Pathway

Abstract Since residual chronic myelogenous leukemia (CML) stem cells may be a cause of relapse after Imatinib (IM) cessation, targeting these IM-resistant stem cells is mandatory for complete cure of this disease. CCAAT/enhancer binding protein β (C/EBPβ), a leucine zipper transcription factor, promotes both cell cycle progression and differentiation toward granulocytes in hematopoietic stem/progenitor cells under stress conditions such as infection. We have recently reported that C/EBPβ was upregulated in leukemic stem/progenitor cells derived from patients in chronic phase of CML (CP-CML) through STAT5, a major downstream target of BCR-ABL. In CML mouse model, C/EBPβ enhanced exhaustion of CML stem cells through promoting their differentiation (Hayashi Y et al, Leukemia 2013). In spite of the upregulation of C/EBPβ by BCR-ABL, CML stem cells will not be exhausted spontaneously in patients with CP-CMP. Therefore, we hypothesized that quiescent CML stem cells maintain their immature status in the bone marrow niche by suppressing C/EBPβ expression or function induced by BCR-ABL and that induction of C/EBPβ expression in CML stem cells through BCR-ABL-independent pathways may be a novel therapeutic strategy targeting CML stem cells. The aim of this study is to propose a novel therapeutic strategy that can stimulate quiescent CML stem cells with cytokine-STATs signalings and induce their exhaustion through C/EBPβ-mediated differentiation. STATs are family members of molecules which convey signals from various kinds of cytokine receptors to nucleus. In order to investigate whether STAT molecules can induce C/EBPβ expression, we first examined the effects of constitutive active (CA) form of STAT1, STAT3 and STAT5 on C/EBPβ expression in a murine hematopoietic stem cell line, EML cells. Retroviral transduction of CA-STAT5 significantly upregulated C/EBPβ mRNA and protein in EML cells. EML cells begun to differentiate toward CD11b+ myeloid lineage upon introduction of CA-STAT5. CA-STAT1 and CA-STAT3 also upregulated C/EBPβ mRNA when they were retrovirally transduced into EML cells (Figure 1). These results suggest that signaling mediated by various kinds of STATs can upregulate C/EBPβ. Consensus binding sites for STATs were not found in the proximal (∼ 4 kb) promoter region of C/EBPβ and we are currently identifying the cis-regulatory elements responsible for the STATs-dependent activation of C/EBPβ.Figure 1Figure 1. Interferon-α (IFNα) exerts STATs-mediated signaling in hematopoietic stem cells and has been used for therapy of CML. Therefore we investigated the possible involvement of C/EBPβ in efficacy of IFNα in CML treatments. Stimulation of EML cells with 500 U/ml IFNα upregulated C/EBPβ mRNA (Figure 2). Higher levels of phosphorylation of STAT1 than those of STAT3 or STAT5 were observed after stimulation with IFNα, suggesting that STAT1 mediated activation of C/EBPβ was induced by IFNα. As previously reported, C/EBPβ was upregulated in EML cells transduced with BCR-ABL (EMLBCR-ABL). Treatment of EMLBCR-ABL cells with IFNα augmented this effect significantly. IM effectively inhibited phosphorylation of STAT5 and blunted the upregulation of C/EBPβ in EMLBCR-ABL cells. Simultaneous treatment of EMLBCR-ABL cells with IFNα and IM resulted in maintained upregulation of C/EBPβ with increased phosphorylation of STAT1 and decreased phosphorylation of STAT5. These data suggested that IFNα treatment can upregulate C/EBPβ independently of signals mediated by BCR-ABL.Figure 2Figure 2. In conclusion, cytokine-STATs signalings can induce C/EBPβ expression in BCR-ABL+ leukemic cells independently from BCR-ABL/JAK-STAT pathway. Stimulations of dormant CML stem cells with cytokines might be a novel treatment strategy to eliminate these populations, leading to complete cure of CML. We are currently evaluating the in vivo effects of IFNα treatment on CML stem cells in mice models. Disclosures: No relevant conflicts of interest to declare.

Loss Of Major Molecular Response As a Trigger For Restarting Tyrosine Kinase Inhibitor Therapy In CP-CML Patients Who Have Stopped Imatinib After Durable Undetectable Minimal Residual Disease

PurposeMore than half of patients with chronic phase chronic myelogenous leukemia (CP-CML) in complete molecular response (CMR) experience molecular relapse after imatinib discontinuation. We investigated loss of major molecular response (MMR) as a criterion for resuming therapy. Patients and methodsA multicenter observational study (A-STIM, According to STop IMatinib) evaluating MMR persistence was conducted in 80 CP-CML patients who had stopped imatinib after prolonged confirmed CMR (24 months or more). Patients with confirmed CMR with 1 or 2 occasional weak positive samples before study entry were also considered eligible. CMR was defined as undetectable BCR-ABL transcript with a sensitivity of at least 40000 amplified copies of the ABL control gene, in accordance with the level of sensitivity routinely applied within laboratories participating in the French GBMHM Network. ResultsMedian time from imatinib initiation to discontinuation was 79 months (range 30-145), median duration of CMR before imatinib discontinuation was 41 months (24-96), and median follow-up after discontinuation was 31 months (8-92). Twenty-nine patients (36%) lost MMR after a median of 4 months off-therapy (2-17). Cumulative incidence of MMR loss was estimated as 35% (95% CI, 25%-46%) at 12 months and 36% (95% CI, 26%-47%) at 24 months whereas probability of losing CMR was estimated as 51% at 12 months (95% CI, 41%-63%) and 54% at 24 months (95% CI, 44%-66%). Fivety two percent of the patients met the criteria for cCMR but experienced occasional BCR-ABL positivity (unstable cCMR). Those patients were not at higher risk of losing MMR as compared to patients with stable cCMR. This observation may potentially increase by two-fold the number of patients eligible for TKI discontinuation. Fluctuation of BCR-ABLtranscript levels below the MMR threshold (≥ 2 consecutive positive values) were observed in 31% of patients after imatinib discontinuation. Using cell sorting in three fluctuating patients, we were able to confirm that BCR-ABL signal was mostly present in the CD15 positive fraction, demonstrating first that BCR-ABL residual signal was not related to long living lymphoid cells and second that residual CML cells retain a clonogenic potential. Treatment-free remission was estimated as 64% (95% CI, 54%-75%) at 12 and 24 months and 61% (95% CI, 51%-73%) at 36 months. Treatment was resumed in 31 patients after loss of MMR. Twenty-three patients regained CMR4.5 and 8 patients are in MMR under therapy after 2+ to 17+ months. Median to time to a second CMR was estimated as 7.3 months in retreated patients. ConclusionThe probability of losing MMR after imatinib discontinuation was estimated as 36% in the long-term. Loss of MMR is a practical and safe criterion for restarting therapy in CML patients with prolonged CMR and could be used for future discontinuation studies. Disclosures:Rousselot:Novartis: Research Funding; BMS: Research Funding. Cony-Makhoul:BMS: Honoraria; Novartis: Research Funding. Nicolini:BMS, Teva, Ariad, Pfizer, Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Etienne:Novartis: Consultancy; Pfeizer: Consultancy; Novartis: Honoraria; BMS: Honoraria. Guerci-Bresler:Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Turhan:BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Novartis: Research Funding. Guilhot:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; BMS: Research Funding; Novartis: Research Funding. Mahon:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Brisol Myers Squibb: Consultancy, Honoraria; Ariad: Honoraria; Pfizer: Honoraria.

Poor Response To Imatinib In Patients With Secondary Chronic Myelogenous Leukemia

IntroductionHigh dose ionizing radiation and chemotherapy, particularly alkylating agents and topoisomerase II inhibitors, are well known causes of secondary acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Similarly, radiation exposure has been linked to chronic myelogenous leukemia (CML) in large epidemiologic studies of atomic bomb survivors and patients who received high-dose radiation for ankylosing spondylitis and cervical cancer. Patients with secondary AML have poorer survival outcomes compared to de novo AML patients. Little information exists on outcomes of patients with secondary CML. In our study, we investigated treatment responses in patients with secondary CML who have been treated with tyrosine kinase inhibitors (TKIs). Study DesignEight patients who developed CML after receiving chemotherapy, radiation therapy or both were identified between three academic institutions. Retrospective chart review was performed, and information was collected regarding the clinical characteristics and treatment of the patients' first malignancies. Data about the patients' CML was also gathered, including time between previous malignancy treatment and diagnosis of CML and type of CML treatment. Finally, hematologic, cytogenetic, and molecular responses to treatment were assessed. ResultsThe mean age of our patients at diagnosis of CML was 58. Three patients were previously diagnosed with breast cancer, two with colorectal cancer, one with peripheral T-cell lymphoma, one with chronic lymphocytic leukemia, and one with Waldenstrom's macroglobulinemia. One of the patients with colorectal cancer also had a history of cervical cancer, for which she received radiation therapy. Six of eight patients (75%) received radiation therapy prior to onset of CML, and six of eight patients (75%) had previously received chemotherapy, two of whom received alkylating agents. On average, patients developed CML eight years after receiving treatment for the previous malignancy (range 3 to 18 years). All of the patients were diagnosed in chronic-phase CML. Based on the Sokal score, three patients (37.5%) had low risk CML, two (25%) had intermediate risk CML, one (12.5%) had high risk CML, and the Sokal score was unable to be calculated in two (25%) patients due to limited information.Six of the eight patients (75%) received imatinib as initial therapy. Only two of these six patients (33%) achieved a complete cytogenetic response (CCyR). The patient who received up-front nilotinib was the only patient who developed molecularly undetectable leukemia to initial therapy. Seven of the eight patients (87.5%) were alive at a mean four year follow-up. One patient had CML with additional cytogenetic abnormalities at diagnosis and progressed to blast-phase. DiscussionThis case series is the first study to specifically examine outcomes in secondary CML patients treated with TKIs. The data raise questions regarding the efficacy of imatinib in patients with secondary CML. Only two of the six patients who received imatinib achieved a CCyR. This is a significantly lower response rate than would be expected in de novo CML. This study suggests a role for the newer tyrosine kinase inhibitors as initial therapy in patients with secondary CML. Larger observational studies are needed to confirm these findings. Disclosures:No relevant conflicts of interest to declare.

Quantitative Monitoring Of Wilms' Tumor 1 Expression In Predicting Relapse After Allogeneic Hematopoietic Stem Cell Transplantation In Patients With Acute Leukemia and Myelodysplastic Syndrome

Relapse is the major cause of treatment failure after allogeneic hematopoietic cell transplantation (HCT) for leukemia and myelodysplastic syndrome (MDS). In order to improve the outcome of leukemia patients after allogeneic HCT, it is imperative to identify reliable markers to predict impending relapse. Wilms' tumor antigen (WT1) is overexpressed in the majority of leukemia and MDS patients and is being considered as a possible universal diagnostic marker for minimal residual disease (MRD), especially since no chromosomal translocation has nearly the frequency of association as WT1 does with leukemia.In this study we prospectively evaluated the prognostic value of MRD monitoring by qRT-PCR for WT1 transcripts. WT1 transcript levels in peripheral blood mononuclear cells (PBMC) were measured utilizing SYBR-Green qRT-PCR on the ABI7300 instrument (Applied Biosystems, Carlsbad, CA) and results were expressed as a ratio of WT1/c-ABL transcript copies normalized by 104 (WT1 ratio: WT1/c-ABLx104). PBMC samples were obtained monthly for 6 months post-HCT, then on alternating monthly schedule until 3 years post-HCT. Patients >18y.o. with confirmed diagnosis of MDS with <20% blasts, AML/ALL in 1st or 2ndCR, and CML in chronic phase undergoing HCT were eligible for the study.A total of 83 patients (median age: 54, range: 19-74) with AML (n=39), ALL (n=24), MDS (n=17), or CML (n=3) received allogeneic HCT after fully ablative (n=39) or reduced-intensity (n=44) conditioning. Donor sources were matched related (n=33), unrelated (n=50), or umbilical cord blood (n=2). Fifty-one patients were considered low-risk (AML/ALL in CR1, CML in 1stchronic phase, or MDS-RA/RARS subtypes) while the remaining 32 patients were considered high risk.Sixteen of 83 patients relapsed with a median time of 238 days post-HCT (range: 76-747). The minimum WT1 ratio that gave specificity of 100% in predicting relapse was 50 (95% binomial exact CI: 92.5-100%), as none of the non-relapsed patients crossed this level. Of 16 patients who relapsed, 12 crossed the WT1 ratio of 50, providing a sensitivity of 75% (95% binomial exact CI: 48- 93%). The positive predictive value (PPV) and the negative predictive value (NPV) performance parameters for the WT1 ratio of 50 were 100% and 94.4%, respectively (Table 1). There was an average number of 63 days (SD=29.3) from crossing the WT1 ratio of 50 to hematologic relapse for the 12 relapsed patients. Since PBMC samples from healthy donors consistently demonstrated a WT1 ratio <10, we also examined different WT1 cutoff ratios (10 to 50) for their performance characteristics (Table 1). Compared with the WT1 ratio of 50, a cutoff ratio at 20 resulted in an increased sensitivity (87.5%) for relapse prediction and days to relapse (78 days), while the specificity and PPV decreased to 85% and 58.3%, respectively. The performance of the cutoff ratio of 20 was improved on PPV (69%) and days to relapse (85 days) in a subgroup of patients with high-risk disease while maintaining good sensitivity and specificity above 80%. Univariate analysis showed WT1 ratios (as a continuous variable), crossing the WT1 ratio of 20 (as a time-dependent variable), high risk disease, and donor age were significantly associated with relapse. In multivariate analysis, crossing the WT1 ratio of 20 remained the only significant factor predicting relapse (HR 56.9 [18-189], p<0.0001).In summary, our data demonstrate that the quantitative measurement of WT1 transcripts is a reliable marker to assess MRD post-HCT for leukemia patients, and its real-time prospective monitoring provides a 2-3 month window of opportunity to introduce medical/immunologic interventions prior to overt hematologic relapse.Table 2WT1 ratio Specificity Sensitivity Positive Predictive Value Negative Predictive Value Days to Relapse (SD)50100%75%100%94.4%63 days (29)4095.5%75%80%94.1%66.9 days (29)3094%75%75%94%71.5 days (30)2085%87.5%58.3%96.6%78 days (28)1056.7%93.8%34.1%97.4%107.5 days (58) Disclosures:No relevant conflicts of interest to declare.

Deep Molecular Responses In Patients With Newly Diagnosed Chronic Myeloid Leukemia Receiving Nilotinib As Assessed Within The EUTOS Laboratory Network In The ENEST1st Study

Deep Molecular Responses In Patients With Newly Diagnosed Chronic Myeloid Leukemia Receiving Nilotinib As Assessed Within The EUTOS Laboratory Network In The ENEST1st Study

A T315I Gate Keeper Mutation Responding To Pegylated Interferon Alfa-2a (Peg-IFN) Monotherapy With Major Molecular Response (MMR4) At 12 Months In An Imatinib-Resistant Chronic Myeloid Leukemia (CML) Patient

Since the IRIS Study in 2000, tyrosine kinase inhibitors (TKI) have become a standard therapy for CML patients in lieu of interferon based therapy. Nevertheless, resistance occurs in roughly 30% of imatinib-treated patients. This resistance is attributable to ABL kinase domain mutations in 50% of cases, in which T315I mutation accounts for 10 to 15%. This latter is resistant to all TKIs available except for ponatinib which enables 75% of complete cytogenetic response (CCyR) in this setting (NEJM November 29, 2012). Before Ponatinib, various strategies were proposed to overcome the T315I mutation including more importantly ‘non-targeted' therapy such as Omacetaxine. IFN is also a ‘non-targeted therapy' known to have a well-established immunological effect. There are also clues about its cellular effects: microarray analyses have shown that IFN alfa can upregulate genes that encode for apoptotic proteins (i.e TRAIL, Fas, caspases 4 and 8 and XAF-1…) conferring the anti-growth effect different from ATP competition specific to TKIs.We report here an 80 years-old imatinib-resistant CML patient harboring the T315I mutation who has been successfully treated with Peg-IFN (Pegasys).His past medical history involved hypertension, asthma and chronic obstructive pulmonary disease. He was diagnosed in July 2008 with a chronic phase CML when he presented with weight loss, splenomegaly, leukocytosis (WBC 80G/l), slight anemia (Hemoglobin 112G/l) and normal Platelet count 240G/L. Karyotyping of 26 metaphase cells in the bone marrow showed Philadelphia (Ph1) chromosome without any additional abnormality. The molecular analysis by real time PCR (Light Cycler 480) detected a BCR-ABL p210 transcript. The Sokal score was high (at 1.24) while the Eutos score was low (31).He started Imatinib 400mg daily in July 2008 and achieved 5% IS BCR-ABL ratio at 3 months and complete cytogenetic response (CCyR) at 12 months. But the patient had never reached the major molecular response (MMR) and ended up losing the cytogenetic response in February 2012 (at 40 months) with an increase of 1.5log in the BCR-ABL ratio. Sequencing analysis for ABL mutation was carried out using the applied biosystems 3500 gentic analyzer. It revealed the T315I mutation at 20%. After a request for Ponatinib was dismissed by our health authorities, we decided to treat the patient with Peg-IFN in June 2012 at the dose of 90 µg per week. At this point, the imatinib had been stopped for 3 months, 8 metaphase cells out of 20 showed Ph1 chromosome without additional abnormality, BCR-ABL ratio was 3% IS, the mutational analysis showed 50% of T315I and 20% of another acquired E255K mutation. But the patient was still in complete haematologic response. figure 1 highlights the decline in the BCR-ABL ratio with MMR reached for the first time at 9 months (from Peg-IFN treatment). Of note, the CCyR was obtained again at this landmark. At 12 months from the Peg-IFN the CCyR was confirmed, the BCR-ABL ratio came very close to MMR4 (0.02% IS) while both T315I and E255K mutations disappeared on mutational analysis. [Display omitted] No side effect was reported except for weight loss when the dose of Peg-IFN was increased to 180 µg per week which was the dose suggested by J.H. Lipton (Leukemia and Lymphoma, March 2007). The average dose in the past 12 months was 90 µg per week. The latter dose is well tolerated and is still ongoing.This observation shows that, though we are in the ponatinib era, there still is a room for interferon, in the management of CP CML patients even in BCR-ABL domain mutated patients because of its different mechanism of action. In this case the Pegylated Interferon a-2-a at the dose of 90µg per week has allowed clearance of both T315I and E255K mutations with a response close to MMR4. Taking into account the slope of the BCR-ABL decline, deeper molecular response is expected before the ASH meeting in December 2013. Disclosures:No relevant conflicts of interest to declare.

Disruption Of Ikaros In Patients' Chronic Phase CML Cells Induces The Features Of Accelerated Phase Disease

Accelerated phase (AP) CML is marked by a rising leucocyte count in the face of maintenance treatment, exaggerated basophilia and accumulating blasts. However, AP is typically short-lived and rapidly succeeded by blast crisis. Genetic alterations resulting in loss of IKAROS activity are associated with the development of both BCR-ABL1+ myeloid and lymphoid leukemias, as well as the related BCR-ABL1-negative MPNs. However, how loss of IKAROS functionality contributes to the malignant features of these leukemias is not clear. Here we demonstrate that direct suppression of IKAROS activity in CD34+ cells from chronic phase (CP) CML patients leads to their rapid acquisition of features of AP without causing progression to acute leukemia.To suppress IKAROS activity in CD34+ CP CML cells, we transduced samples from 4 patients with a lentiviral GFP vector encoding a dominant-negative isoform of IKAROS (dnIK6), or an empty control YFP vector, and compared their progeny generated in cultures containing mouse stromal cells engineered to produce human SCF, IL-3, FLT3L and G-CSF. In all cases, we found dnIK6 transduction caused a rapid (evident within 2 weeks) and sustained (for at least 6 weeks) increase in total cell output (4-20x higher than YFP+ controls at 4-6 weeks, p<0.01). More detailed analysis showed that both populations of transduced cells produced similar numbers of monocytes and neutrophils and the increased cell output from the dnIK6-transduced cells was largely due to a consistent 50-200x increase in basophil production (with occasional increased eosinophils also), quantified using stained cytospins, and confirmed by flow cytometry (presence of CD34-33+117-14-15-HLA-DR-25+9+ cells). Similarly, comparison by qRT-PCR of dnIK6 versus control nonadherent cells present after 2-4 weeks showed 10-50-fold lower MPO, 2-9-fold higher HDC and 20-65-fold higher PRG2 transcripts. Notably, the cloning efficiency of the initially transduced control and dnIK6-transduced CD34+ CML cells in both semi-solid media and suspension culture was not significantly different. However, the proportion of dying/dead mature (nonadherent) cells (as measured by propidium iodide staining) derived from control cells was ∼30x higher than from the dnIK6-transduced cells (4.9±1.0% vs 0.15±0.03%, p=0.002). This suggested a dnIK6-augmented survival of terminally differentiating cells causing an accumulation of their mature progeny. In addition, assessment of 6-week cultures revealed a strikingly higher number of dnIK6-derived CD34+ cells and colony-forming cells as compared to controls (49±15-fold and 19±6-fold higher, respectively).To interrogate the molecular mechanisms underlying the dnIK6 effects on cell output, we compared the levels of activated signaling proteins by intracellular flow cytometry. This showed increased activation of STAT5 (80±14% higher median fluorescent intensity (MFI), p<0.001), MAPK (61±11% higher MFI, p=0.01) and β-catenin (44±9% higher MFI, p=0.02) in the CD34+ progeny of dnIK6-transduced CP CML cells. In addition, qRT-PCR analysis showed transcripts encoding key repressors of JAK-STAT (SOCS2, SH2B3) and β-catenin signaling (including GSK3B, AXIN1 and CK1) were suppressed in dnIK6-transduced CD34+ cells by comparison to CD34+ derivatives of control-transduced cells from the same patients. After a total of 12 weeks in culture, the control-transduced CML cells had largely disappeared, whereas the population produced by the dnIK6-transduced cells continued to expand, with a mean cumulative cell output after 12 weeks of 6x106 cells from 104 starting CD34+ cells (vs 2x105 from controls) along with a marked increase in CD34+ cells, rising from 1-3% at week 6, to 12-60% at week 12. However, plating of these CD34+ cells in semi-solid media yielded basophil or monocyte but not blast colonies, demonstrating that differentiation was not strongly impaired.In summary, IKAROS disruption in patients' CP CD34+ CML cells recapitulates cardinal features of AP disease, including an increased cell output associated with an enhanced production of basophils and an eventual accumulation of primitive cells. We further identify dnIK6-mediated suppression of SOCS2 and the β-catenin destruction complex, with enhanced activation of STAT5 and β-catenin in progenitor cells, as a likely key mechanism contributing to the altered biology of AP cells. Disclosures:No relevant conflicts of interest to declare.

Activation Of p53 By MDM2-Inhibition Sensitizes Quiescent Blast Crisis CML Stem/Progenitor Cells To Bcl-2 Inhibitor- and Tyrosine Kinase Inhibitor-Induced Cell Death

The transcriptional activation of pro-apoptotic Bcl-2 family members is a major mechanism of p53-mediated cell death. p53 can also directly contribute to mitochondrial mediated apoptosis by interacting with Bcl-2 family proteins. A number of anti-apoptotic Bcl-2 proteins such as Mcl-1 and Bcl-xL are regulated by the Bcr-Abl tyrosine kinase and confer apoptosis resistance to CML cells. It is therefore not surprising that p53 gene alterations may play a role in the disease progression of CML. The reported p53 mutation rate of 30% suggests potential therapeutic benefit of p53 activation by MDM2 inhibition alone or as sensitization of CML to therapeutic agents.Tyrosine kinase inhibitors (TKIs), the front-line therapy for patients with chronic phase CML, are less effective in patients with blast crisis (BC) CML and inactive against quiescent CML stem/progenitor cells. Here we examine the effects of nutlin3a, which activates p53 by inhibiting MDM2, and its combinations with the Bcl-2 inhibitor ABT-737 and the TKI nilotinib on the viability of proliferating and quiescent CD34+CML stem/progenitor cells obtained from patients with BC CML.Mononuclear cells from patients with BC CML who were resistant to multiple TKIs were stained with the cell division tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM) derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells are distinguishable by flow cytometry (within 7 to 12 days), the cells were treated with nutlin3a, ABT-737, nilotinib, nutlin3a plus ABT-737, or nutlin3a plus nilotinib for 48 hours with or without MSC co-culture. The cells were then stained with a CD34 antibody, and apoptosis (annexin V staining) was determined by flow cytometry. Apoptosis in proliferating and quiescent progenitor cells was defined as percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbrightcells, respectively.Nutlin3a alone was able to decrease viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50 = 2.50 ± 0.92 μM and 2.54 ± 0.60 μM, respectively) and quiescent (IC50 = 3.70 ± 1.22 μM and 4.27 ± 0.77 μM, respectively) CD34+ CML stem/progenitor cells. Although not very active by itself, ABT-737, when combined with nutlin3a (n = 6), induced apoptosis synergistically in proliferating (CI = 0.24 ± 0.12) and more so in quiescent (CI = 0.09 ± 0.10) CD34+CML stem/progenitor cells, even when cells were co-cultured with MSCs (CI = 0.22 ± 0.09 for proliferating and CI = 0.03 ± 0.05 for quiescent stem/progenitor cells). The combination of nutlin3a with nilotinib (n = 4) was also highly synergistic in proliferating stem/progenitors (CI = 0.02 ± 0.04 without and CI = 0.05 ± 0.04 with co-culture) and in quiescent progenitors (CI = 0.07 ± 0.29 without and CI = 0.03 ± 0.06 with co-culture).Treatment of primary CML blast cells with nultin3a increased p53, and pro-apoptotic Bax and Puma levels in all samples examined, indicating activation of transcriptional activity of p53. In addition, nutlin3a treatment decreased the protein levels of anti-apoptotic Bcl-xL and/or Mcl-1. Nilotinib also decreased the expression, both at the RNA and protein levels, of Mcl-1 and Bcl-xL, even in patients who had not responded to nilotinib treatment clinically. ConclusionsHere we demonstrate that activation of p53 induces apoptosis in proliferating and quiescent BC CML stem/progenitor cells and sensitizes to Bcl-2 inhibitor- and TKI-induced cell death by, at least in part, regulating the expression of Bcl-2 family proteins. Results suggest a potential for utilizing this strategy in the treatment of BC CML, and also for the elimination of quiescent CML stem/progenitor cells. Disclosures:No relevant conflicts of interest to declare.

Targeting β-arrestin2 Enhances Survival in a Murine Model of Chronic Myeloid Leukemia

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative neoplasm of hematopoietic stem cells characterized by presence of a dysregulated BCR-ABL fusion protein which leads to constitutive activation of tyrosine kinase activity. Classically, CML is treated with tyrosine kinase inhibitors (TKIs). However, TKI therapy is non-curative, and persistence of quiescent leukemia stem cells likely accounts for the inability of these agents to cure CML. β-arrestins are multifunctional adapter proteins which regulate G protein-coupled receptor (GPCR) signaling and trafficking and have recently been identified as mediators of distinct cellular signaling cascades independent of G proteins. β-arrestins also play a role in the Smoothened/Hedgehog pathway, as well as in the Wingless/Frizzled (Wnt/Fz) signaling axis, both of which have been associated with the development of CML. We, therefore, hypothesize that β-arrestin2 (βarr2) is necessary for the development and propagation of CML and may function as a therapeutic target. AimDemonstrate that loss of βarr2, using an inducible conditional knockout mouse model, slows progression of CML. MethodsWe used a standard murine retroviral transduction system to model chronic phase BCR-ABL positive CML. KLS cells (Lin-, Sca-1+, c-kit+) were harvested from bone marrow of donor C57BL6/J βarr2F/F-CreERT2+/- (Cre positive, CD45.2) and age matched C57BL6/J βarr2F/F-CreERT2-/- (Cre negative, CD54.2) male mice. KLS cells were retrovirally transduced with MSCV-BCR-ABL-IRES-GFP and were subsequently injected retro-orbitally into sublethally irradiated congenic wild type recipient male mice (CD45.1). Donor mice were engineered using global Cre-ER/loxP technology in order to induce site-specific recombination and loss of βarr2 when treated with tamoxifen. Mice who received Cre positive cells lost βarr2 only in hematopoietic cells. Recipient mice were treated with tamoxifen 75mg/kg daily via intraperitoneal injection for 5 days starting day 3 after transplant and were monitored for signs of leukemia development. Weekly blood analysis included WBC count, number of donor cells by flow cytometry, blood film, and qPCR for BCR-ABL expression. Survival was compared between animals receiving Cre positive (βarr2F/F-CreERT2+/-) and Cre negative (βarr2F/F-CreERT2-/-) donor cells as well as both tamoxifen treated and untreated conditions. ResultsTreatment of donor C57BL6/J βarr2F/F-CreERT2+/- mice with tamoxifen resulted in decreased βarr2 expression within 10 days in multiple tissues including spleen and bone marrow. By day 10, βarr2 expression was 9.5 ± 3.0% in Cre positive mice relative to pretreatment expression levels (Figure 1).In total, 8 mice received Cre positive cells and 11 mice received Cre negative cells. Ten of 11 (90.9%) Cre negative mice developed CML as evidenced by splenomegaly, leukocytosis, increased BCR-ABL expression measured by qPCR and increased number of donor cells detectable by flow cytometry. Median survival was 15 days. Six of 8 (75%) Cre positive mice developed disease with median survival of 27 days (HR 3.2, 95% CI; 1.525-12.2, p=0.013)(Figure 2). At day 11, flow cytometry for donor CD45.2 cells present in peripheral blood of recipient mice was 38 ± 2.05% in Cre negative versus 16.5± 3.9% in Cre positive mice (p<0.0001). Spleen size at death and WBC count at day 11 were not significantly different between groups. ConclusionsThese data demonstrate that targeting βarr2 prolongs the course of disease in chronic phase CML and raise the possibility that loss of βarr2 after disease onset may lead to disease regression. βarr2 may therefore represent an alternative therapeutic target for CML independent of tyrosine kinase inhibition. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

P53 and c-Myc Are Critical Signaling Hubs That Maintain Chronic Myeloid Leukemia

Abstract Chronic myeloid leukemia (CML) is a clonal disorder of the hematopoietic system, leading to increased production of mature and progenitor myeloid cells. Although protein tyrosine kinase inhibitors (TKI) have been successful in managing the disease, there are exceptions where drug resistance and onset of blast crisis occur. Furthermore TKIs are ineffective against leukemic stem cells (LSC) that are responsible for disease initiation and maintenance. We have shown mRNA changes in primitive hematopoietic cells do not correlate directly to protein changes. Therefore to elucidate fundamental cellular differences between CML and normal cells we employed a proteomic approach (mass spectrometry with isobaric tagging for relative quantification). This approach permits unbiased analyses using direct comparative quantification of peptides and thus proteins from chronic phase CML and normal CD34+ human samples. Systematic data analysis identified that the majority of deregulated proteins are connected and regulated by two oncogenes with well defined roles in human disease, p53 and c-myc. The direction of regulation inferred suppression of p53 and up-regulation of c-myc. Altered expression of key proteins was validated using western blotting and immuno-fluorescence approaches. All (6/6) candidate/hub proteins identified using mass spectrometry were confirmed using these orthogonal approaches. Based on our systematic analysis, we targeted the candidate hubs using the drugs RITA (activates p53) and CPI-203 (inhibits c-myc expression; provided by Constellation Pharmaceuticals). In CML CD34+ cells, RITA reduced cell expansion in a concentration-dependent manner and induced significant levels of apoptosis as confirmed by positive staining of Annexin V and 4',6-diamidino-2-phenylindole (DAPI) using flow cytometry. CPI-203 also reduced cell expansion, but importantly induced differentiation in addition to apoptosis, as supported by flow cytometric monitoring of levels of carboxyfluorescein succinimidyl ester (CFSE) and CD34. Overlays of CFSE plots for untreated control vs. CPI-203 demonstrated that as cells divided in the presence of CPI-203, there was clear and rapid loss of CD34 expression which was not seen with RITA treatment. By measuring the dose-effect relationship of each drug alone and in combination, we demonstrated potent synergy with combination index (CI) values ranging from 0.034-0.286 based on loss of cell viability. Using flow cytometry we gated on CD34+38- CML cells to enable quantification of the differential effects of each drug alone and in combination against the most primitive and quiescent 1-5% of total CD34+ cells. Critically the apoptotic effect was inclusive of primitive CD34+38- cells and quiescent CFSEmax populations. In addition, experiments combining RITA and CPI-203 demonstrated undetectable colony forming cell units at the highest concentrations of drug used. Importantly, it appears that combining these two drugs has negligible effects on normal CD34+ cell counts, apoptosis and CFSE profiles. Currently NOD-SCID IL2R gamma null (NSG) repopulation assays are underway to determine if these drugs affect stem cells capable of engrafting immunocompromised mice. Our systems biology approach suggests that altered c-myc and p53 function underlie the most significant cellular differences within CML CD34+ cells, which has not been previously demonstrated. We confirm that in CML, p53 and c-myc hub proteins have the ability to modulate downstream defined target proteins thereby enhancing survival and proliferation and thus allowing maintenance of disease. Disclosures: No relevant conflicts of interest to declare.

Expanded Access Program Of Ponatinib (AP24534) For Patients (Pts) With Refractory Chronic Myeloid Leukemia (CML) Or Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL)

BackgroundPonatinib is a pan-BCR-ABL inhibitor with activity against BCR-ABL, including the TKI resistant T315I and all other clinically relevant mutant. The efficacy and safety of ponatinib (45 mg orally once daily) in pts with CML in chronic (CP-CML), accelerated (AP-CML), or blastic (BP-CML) phase, or Ph+ ALL were evaluated in an expanded access program. Methods49 pts, including 24 CP-CML, 5 AP-CML, 8 BP-CML (100% Myeloid Blasts), and 12 Ph+ ALL were registered at 2 institutions. Pts were resistant or intolerant (R/I) to dasatinib or nilotinib, or had the T315I mutation at baseline. For this analysis, pts were grouped in cohorts according to the disease stage and presence or not of T315I. ResultsThe median age for CP-CML, AP-CML, BP-CML, and Ph+ ALL pts was 44, 36, 56, and 57 yrs, respectively. Median yrs from diagnosis to start of ponatinib were 7, 1, 3 and 2, respectively. 94% had received imatinib, 80% dasatinib, 59% nilotinib, 2% bosutinib, and 24% had undergone prior SCT. Among 37 pts evaluable for response to prior dasatinib or nilotinib, 84% had history of resistance to these agents, 16% were intolerant. 92% pts had received at least 2 prior TKI, and 45% had received at least 3. Reported response rates with the most recent TKI were 63% (15/24) MCyR for CP-CML, and CHR 60% AP-CML, 0% BP-CML, 33% Ph+ ALL. After a follow-up of 9 months on therapy, 49% of CP-CML, 60% of AP-CML, 25% of BP-CML, and 9% of Ph+ ALL pts remain on study. The reasons for discontinuation were progressive disease (38%, 40%, 75%, and 83%, respectively) and adverse events (13%, 0%, 0% and 8%, respectively). Among the 49 pts, 58% (14/24) pts in CP-CML have achieved MCyR (46% CCyR). 60% (3/5) AP-CML achieved MCyR (2 of them CCyR), and 35% (7/20) BP/Ph+ALL achieved MCyR (5 of them CCyR). MMR is available by IS only for 40 pts and 26% achieved MMR, and at 9 mos. MMR was achieved by 21% CP, 0% in AP, 0% in BP-CML and 38% Ph+ ALL. The median PFS for all pts was 5.8 mos. Estimated PFS at 9 mos. was 50% overall, and 71% in CP, and 75% in AP, 25% in BP-CML and 17% in Ph+ ALL. In CP-CML, AP- CML, BP-CML and Ph+ALL the median PFS was estimated as 12.5 mos., 13.3 mos., 3.5 mos. and 1.4 mos. respectively. Overall median OS was not reached. The probability of OS at 9 mos. for all pts was 80%.; in CP-CML it was 100%, in AP-CML was 75%, in BP-CML 58% and in Ph+ ALL was 53% at 9 mos. The most common AEs were thrombocytopenia (18%), G1-2 rash (14%), and neutropenia (12%) and the G3-4 AEs were thrombocytopenia (4%) and pancreatitis (6%) with G3-4 lipase elevation 8% and G3 amylase 8%. ConclusionsPonatinib has clinical activity in refractory CML and pretreated Ph+ leukemia patients who have limited treatment options. The results of this expanded access study match those of the pivotal PACE trial confirming the clinical benefit with ponatinib in this setting.Table 1Overall results based on primary endpoint for this analysisCP-CMLAP-CMLBP-CML/Ph+ ALLMCyRCHRCHRResponse n/N% (95% CI)Response n/N% (95% CI)Response n/N% (95% CI)R/I10/1567 (41.34, 84.80)2/450 (14.66, 85.34)2/1020 (6.02, 51.78)T351I5/956 (26.23, 81.29)1/1100 (15.81, 98.74)2/1020 (6.02, 51.78)Total15/2463 (42.52, 78.87)3/560 (22.28, 88.19 )4/2020 (8.21, 41.9) Disclosures:Kantarjian:ARIAD: Research Funding. Jabbour:Ariad, BMS, Novartis, and Pfizer: Consultancy. O'Brien:ARIAD: Research Funding. Pinilla:Ariad: Advisory Board Other, Research Funding, Speakers Bureau. Cortes:Ariad, Pfizer and Teva: Consultancy; Ariad, BMS, Novartis, Pfizer, Teva: Research Funding.

Blast Crisis CML Cells Require IFN-γ Conditioning For Effective GVL Whereas Chronic Phase CML Cells Do Not: An Explanation For Chronic Phase CML GVL-Sensitivity

While the GVL effect is potent against chronic phase (CP) CML, it is less effective against blast crisis (BC) CML and AML. To better understand this difference we have been studying GVL against mouse models of CP-CML (mCP-CML) and BC-CML (mBC-CML) induced by infecting bone marrow (BM) with retrovirus encoding bcr-abl alone (mCP-CML) or also with a retrovirus encoding the NUP98-HOXA9 fusion (mBC-CML). We previously reported MHCII-/- mBC-CML and mCP-CML to be completely resistant to CD4-mediated GVL, but the leukemia stem cells (LSCs) of each expressed little if any MHCII. We hypothesized that MHCII might be upregulated in an alloimmune environment. Consistent with this, in mice transplanted with allogeneic T cells, mBC-CML and mCP-CML LSCs upregulated MHCII as well as MHCI and ICAM-1. To test the hypothesis that IFN-γ upregulated LSC MHC post BMT, we created IFN-γR-/- mBC- and mCP-CML. IFN-γR-/- mBC-CML LSCs did not upregulate MHCII, MHCI or ICAM-1 in vivo, whereas they were upregulated on IFN-γR-/- mCP-CML LSCs. Consistent with this, IFN-γR-/- mBC-CML was completely resistant to GVL (C3H.SW→B6) mediated by CD4 or CD8 cells whereas GVL against IFN-γR-/- mCP-CML was equivalent to that against wt mCP-CML. Therefore independence from IFN-γ conditioning is a key property that at least in part explains the sensitivity of mCP-CML to GVL. In contrast, type I IFN receptor-deficient mCP-CML and mBC-CML upregulated MHC and were fully GVL-sensitive. That IFN-γR-/- mBC-CML was resistant to CD8-mediated GVL was surprising given that mBC-CML LSCs are constitutively MHCI+. These data suggest that IFN-γ does more than simply upregulate MHCI. To further explore this we performed gene expression analysis. Illumina arrays revealed that wt mBC-CML and not IFN-γR-/- LSCs from alloimmune mice have higher mRNA levels of death pathway genes Ripk1, Fas, Caspases, 1,2,8, Diablo, Apaf1 and Mdm2 and antigen presentation genes Ciita, Tap1, and proteasome subunit Psmb9, in addition to the canonical IFN-γ-induced gene IRF1. In contrast, c-myc expression was reduced. We next focused on the effects of IFN-γ on mBC-CML LSCs. MHC upregulation did not require that T cells make TCR:MHC interactions as CD8 cells induced upregulation of MHCII in MHCI-/- mBC-CML and CD4 cells induced MHCI upregulation on MHCII-/- mBC-CML. MHCII+ mBC-CML were bona fide LSCs as they transferred mBC-CML, but the resultant leukemias were MHCII-, indicating that MHCII upregulation was dynamic. In vitro IFN-γ led to STAT1 phosphorylation within 15 minutes whereas MHCII upregulation took 24-48 hours. Upregulation could be initiated with an IFN-g pulse as short as 30' but was maximal with a 24 hour stimulation. Donor T cells were the key source of IFN-γ as IFN-γ-/- T cells failed to induce MHCII upregulation and did not mediate GVL, despite engraftment and expansion. Given that cognate interactions were not required for IFN-γ-induced MHCII upregulation, we tested whether GVL could be augmented by IFN-γ-producing T cells that could not directly kill LSCs. To do so, irradiated B6 mice were reconstituted with BALB/c BM, B6 MHCI- mBC-CML without or with BALB/c T cells as follows: a) wt CD4; b) IFN-γ-/- CD4; c) wt CD4 + wt CD8; or d) IFN-γ-/- CD4 + wt CD8. Despite not being able to interact with MHCI- mBC-CML, wt CD8 rescued LSC MHCII upregulation in recipients of IFN-γ-/- CD4 cells and improved early GVL in recipients of both wt and IFN-γ-/- CD4 cells as measured by the number of mBC-CML cells in spleen, blood and BM on day +14. However, the effect was short-lived as later post transplant mBC-CML LSCs lost MHCII expression, likely due to contraction of IFN-γ-producing T cells, and mice succumbed to mBC-CML. These studies suggest that a requirement for IFN-γ stimulation is a key feature that distinguishes GVL against BC-CML and AML from GVL against CP-CML. This could explain why in the clinic a lower intensity alloimmune response is sufficient to contain mCP-CML whereas such responses are generally insufficient against BC-CML and AML. Our work also suggests that T cells that do not recognize leukemia cells can augment GVL. This hypothesis is consistent with a recent report that early CMV reactivation is associated with a lower risk for AML relapse independent from aGVHD (M.L. Green et al,Blood, 2013). These data provide a strong rationale to develop methods for safely delivering IFN-γ to AML and BC-CML cells post alloSCT. Disclosures:No relevant conflicts of interest to declare.

Axl Represents a Therapeutic Target In T315I-Mutated and WT Chronic Myeloid Leukemia

Abstract BCR-ABL1 inhibitors have revolutionized treatment of CML patients. However several drawbacks remain, including therapy resistance of T315I-mutated CML and incapability of current drugs to eliminate quiescent CML stem cells warranting development of novel therapies. In addition, drugs with the potential to enhance efficacy of BCR-ABL1 targeting agents could improve treatment of CML patients. Members of the Tyro3, Axl, Mer receptor (TAMR) tyrosine kinase family are abundantly expressed in physiological and malignant hematopoiesis and their ligand Gas6 can support hematopoietic (progenitor) cells. Evidence in the literature indicates that Axl is upregulated upon treatment with imatinib (IM). In this study, we investigated the relevance of the Gas6-Axl axis in CML patients and the therapeutic potential of the clinically applicable small molecule Axl inhibitor BGB324 (former designation R428) in primary CML (stem cell) samples, cell lines and preclinical models. In a first step we quantified Axl-expressing cells by flow cytometry in chronic phase (CP) CML bone marrow at primary diagnosis and healthy bone marrow donors. Here, we found higher numbers of Axl-positive cells in CML bone marrow compared to controls (11.42±0.42% (n=5) vs. 0.65±0.10% (n=6), respectively; p=0.0015). In addition, we determined Gas6 plasma levels by ELISA in healthy controls and CML patients in CP and blast crisis (BC). These analyses revealed that Gas6 plasma levels were upregulated in a stage specific manner (plasma levels of Gas6: healthy controls 1290±684 pg/ml (n=14), CP 3465±405 pg/ml (n=50), BC 10940±3868 pg/ml (n=7); p=0.0001). Thus, the Gas6-Axl axis represents a potential therapeutic target in CML patients. Based on this finding we analyzed efficacy of BGB324 in Axl-expressing BV173, KCL22, K562, BaF3_BCR-ABL1-wt and BaF3_BCR-ABL1-T315I cell lines in vitro. We found inhibition of proliferation in all analyzed cell lines with IC50 values ranging from 500-3000 nM. Combination experiments with the IC50 dose of BGB324 and IM revealed additive effects of both treatments in all cell lines. Dose finding experiments with BGB324 in sorted CD34+ primary CML cells grown in the presence of physiological growth factors yielded a mean IC50 of 1.1 ± 0.3 mM (n=3), which was similar to nilotinib. BGB324 did not accumulate CD34+CFSEmax (undivided) cells any more than nilotinib but enhanced apoptosis of CD34+ cells in combination with nilotinib. Notably, there was a consistent inhibitory effect of 3 mM BGB324 alone or in combination with nilotinib against colony forming cells (CFC) with the more primitive BFU-E and GEMM being most sensitive to inhibition (n=4). Interestingly, the Ph- lymphocytes (confirmed by FISH) sorted simultaneously with Ph+ CD34+ cells from the same CML patient were unaffected in terms of viability when treated with Axl inhibitor; thus the BGB324’s activity is cell context specific. Encouraged by these data we analyzed efficacy of BGB324 in an aggressive preclinical CML model in which bone marrow cells were retrovirally transduced with constructs containing T315I-mutated BCR-ABL1. Transduced cells were subsequently i.v. transplanted into sublethally irradiated recipient mice, who rapidly developed blast crisis CML. Mice were treated with 25 mg BGB324 or vehicle BID by oral gavage starting from day 3 after transplantation when homing of transduced cells to the bone marrow is completed. In this model we found a significant prolongation of survival upon treatment with BGB324 (Figure 1). Analyses of the leukemia phenotype by differential blood counts and flow cytometry revealed that BGB324 reduced leukemia cell burden (WBC 138±20x103/µl (n=14) vs. 60x103±21 k/µl WBC in the BGB324 treated group (n=13); p=0.0078). Taken together, these data suggest that the Gas6-Axl axis represents a therapeutic target and that BGB324 is a potent molecule effective against T315I-mutated and wt CML alone and in combination with TKI. Furthermore, BGB324 induces apoptosis of quiescent Ph+ CML stem/progenitor cells. Thus BGB324 might open up novel therapeutic avenues in CML patients. Disclosures: Loges: BerGenBio: research support Other.