ALBERT

Description: Introduction: Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by expansion of the granulocytic, erythrocytic, and megakaryocytic lineages in the bone marrow and peripheral blood, and in most cases, by the presence of a JAK2 mutation. Survival of patients with PV is decreased compared with age-matched controls, and this is mainly due to thromboembolic complications followed by progression to post-PV myelofibrosis and acute leukemia. While no curative treatment exists, cytoreductive treatment with hydroxyurea (HU) or ropeginterferon is approved in EU for first-line therapy, and ruxolitinib (RUX) is approved in EU and US for second-line therapy in patients with HU intolerance or resistance. The current futility analysis assesses the efficacy of ruxolitinib in newly-diagnosed PV treated within the Ruxo-BEAT trial. Methods: This clinical trial entitled "Ruxolitinib versus Best Available Therapy in patients with high-risk Polycythemia Vera or high-risk Essential Thrombocythemia" (Ruxo-BEAT; NCT02577926) is a multicenter, open-label, two-arm phase-IIb trial with a target population of 380 pts with PV and ET. Patients in first-line PV and in first and later lines ET are randomized in a 1:1 manner to receive either RUX or best available therapy (BAT). Crossover from BAT to RUX is possible in eligible patients after 6 months. Patients with PV in the RUX arm receive a starting dose of 10 mg bid and may increase their dose up to 20 mg bid. Primary endpoint is the rate of complete clinicohematologic response rate (CHR) at month 6 as defined by Barosi et al Blood 2009. Secondary endpoints include differences in the absence of phlebotomies, spleen size, patient-reported outcomes, and survival. This is a pre-specified futility analysis of RUX in the PV arm, after 50 PV patients had been enrolled. Of the 50 patients, 28 patients with newly-diagnosed PV were randomized into the RUX arm and were analyzed (a maximum of 6 weeks of HU, anagrelide, or interferon therapy was allowed). The PV arm would have to be closed if no favorable trend were observed for RUX for any of the following variables: (1) improvement (decrease) in the hematocrit level during 6 months of treatment, (2) improvement (decrease) of the JAK2V617F allele burden during 6 months of treatment, or (3) improvement of one of the following three symptom variables assessed by physician´s judgement or via MPN Symptom Assessment Form (MPN-SAF) during 6 months of treatment: pruritus, night sweats, or bone pain. Differences between screening (Hct) or baseline (all other variables) and end of month 6 (all variables) were calculated using Fisher´s exact test (for physician-assessed pruritus and night sweats) or the Wilcoxon matched-pairs signed rank test (all other variables). Results: 28 patients received RUX for at least 6 months. After 6 months, the mean hematocrit level decreased from 45.9+/-5.6% to 41.0+/-5.0% (mean+/-SD) (p=0.0003). The number of phlebotomies calculated per year decreased from 4.2+/-3.9% to 0.96+/-2.1 (p=0.0009). Mean JAK2V617F allele burden decreased from 50.2+/28.4% to 44.0+/-28.5% (p=0.0039). The percentage of patients, as assessed by the physician, with pruritus or night sweats decreased from 41% to 26% (trending with p=0.13), and from 30% to 11% (p=0.02), respectively. The points reported by patients themselves on the MPN-SAF survey for pruritus decreased from 2.7+/-3.0 to 1.3+/-1.5 (p=0.0095) and there was a strong trend for reduction of night sweat points (from 3.1+/-3.6 to 1.6+/-2.4; p=0.0579), while the points for bone pain remained unaltered (2.0+/-2.8 to 1.4+/-2.2; p=0.215). Conclusion: Treatment with ruxolitinib in first line PV is efficient regarding the above-mentioned endpoints. Recruitment of our trial will be ongoing. In order not to weaken the study´s statistical power, comparison of both arms was not performed. Disclosures Koschmieder: Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Isfort:Mundipharma: Other: Travel reimbursement; Amgen: Other: Travel reimbursement; Hexal: Other: Travel reimbursement; BMS: Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement; Roche: Other: Travel reimbursement; Alexion: Other: Travel reimbursement. Schafhausen:Novartis: Consultancy, Honoraria; Incyte: Consultancy, Equity Ownership, Honoraria. Griesshammer:Novartis: Consultancy, Honoraria, Speakers Bureau. Platzbecker:Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Döhner:CTI Biopharma: Consultancy, Honoraria; Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Jost:Abbvie: Consultancy, Patents & Royalties: Royalty payments for the drug compound ABT-199, Research Funding; Bohringer: Consultancy, Research Funding; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Celgene: Other: Travel Support; Novartis: Research Funding. von Bubnoff:Novartis: Research Funding. Stegelmann:Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Crysandt:Amgem: Other: travel grant; celgene: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant; Incyte: Membership on an entity's Board of Directors or advisory committees. Gezer:AMGEM: Membership on an entity's Board of Directors or advisory committees. Brümmendorf:Merck: Consultancy; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Research Funding. OffLabel Disclosure: Ruxolitinib as first-line treatment in newly-diagnosed PV

Description: We have previously reported on the efficacy of the JAK1/2 inhibitor ruxolitinib in corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) in 95 patients (pts) (Leukemia 2015;29(10):2062-8). To assess long-term follow-up results, we collected data from the same pts treated in 19 centers in Europe and the US. Pts were classified as SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). Median numbers of pre-ruxolitinib GVHD treatment lines were 3 (1-7) and 3 (1-10) for SR-aGVHD and SR-cGvHD, respectively. The median follow-up was 19 and 24 months for aGVHD and cGVHD, respectively. The 1-year overall survival (OS) from was 62.4% (CI: 49.4%-75.4%) and 92.7% (CI: 84.7%-100%) for SR-aGVHD and SR-cGvHD, respectively. The estimated median OS (50% death) was 18 months for aGVHD and not reached for cGVHD patients. The median duration of ruxolitinib treatment was 5 and 10 months for patients with SR-aGVHD and SR-cGVHD, respectively reflecting the different biology of the diseases. At follow-up, 22/54 (41%) of SR-aGVHD patients and 10/41 (24%) of SR-cGVHD patients have an ongoing response and are free of any immunosuppression. GVHD relapse or progression after achieved PR/CR was observed in 14/45 (31%) and 13/36 (36%) patients with SR-aGVHD and SR-cGVHD, respectively. Response to re-treatment with Ruxolitinib or any immunosupressive therapy was seen in 11/14 (78%) and 11/13 (86%) patients with SR-aGVHD and SR-cGVHD, respectively. Cytopenia (any grade) and CMV-reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. These findings extend our previous report by showing that patients with SR-aGVHD and SR-cGVHD may benefit long-term from ruxolitinib treatment with an OS that is relatively high for steroid-refractory GVHD. GVHD-relapse or GVHD-progression rates were moderate and more than 75% of the relapse/progression patients responded to re-treatment with ruxolitinib or other immunosuppression. Disclosures Meyer: Stanford University: Patents & Royalties. Marks:Pfizer: Honoraria. Lübbert:Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding. Scheid:Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work. Kobbe:Celgene: Honoraria, Other: travel support, Research Funding; Jansen: Honoraria, Other: travel support. Negrin:Stanford University: Patents & Royalties. Brune:Meda Pharma: Consultancy. Mielke:JAZZ Pharma: Speakers Bureau; Novartis: Consultancy; MSD: Consultancy, Other: Travel grants; Gilead: Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Kröger:Sanofi: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Peschel:MophoSys: Honoraria. von Bubnoff:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding.

Description: The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.

Description: Abstract 3247 Poster Board III-1 The tyrosine kinase inhibitor Imatinib is the gold standard in conventional treatment of CML. However, the emergence of resistance to IM remains a major problem. Alternative therapeutic strategies of IM-resistant BCR-ABL positive leukemias are urgently needed. One promising target for anticancer therapeutics is represented by the Aurora kinase family. These serine/threonine kinases are involved in regulating multiple steps of mitosis, including formation of bipolar spindle, chromosome alignment, spindle checkpoint function and cytokinesis. We report on studies accomplished with a small molecule inhibitor AS703569 (Merck Serono), which targets Bcr-Abl and Aurora kinases A-C. We could show that AS703569 exhibited strong anti-proliferative and pro-apoptotic activity against murine BaF3- cells ectopically expressing wild type (wt) or IM-resistant BCR-ABL mutants, including those harbouring the strongly resistant T315I mutation. This effect was observed already at rather low-AS703569 concentrations, at which Aurora- but not the Bcr-Abl kinase was inhibited. Furthermore, in cell cycle analysis we observed cells with a large 4N peak and DNA content more than 4N, indicating extensive polyploidisation, a consequence of continued cell cycle progression in the absence of cell division. Recent studies have revealed that this phenotype is based on suppression of Aurora B kinase activity, indicating that Aurora B inhibition is the major effect of AS703569 in Bcr-Abl positive cells. To confirm this assumption we designed MSCV based retroviruses encoding different point mutations in the Aurora B ATP binding site, which should lead to resistance against AS703569. By this strategy we were able to identify an AS703569 resistant mutant (Aurora B G216V). This mutant shows significant resistance in vitro and is able to augment the antiproliferative capacity of AS703569 in Bcr-Abl positive cells. Taken together, our data demonstrate that anti-proliferative effects of AS703569 in Bcr-Abl positive cells are primarily mediated by functional inhibition of Aurora kinases, especially of Aurora kinase B. Since Aurora kinases are clearly implicated in tumorgenesis, they will become a high potential therapeutic target for anticancer therapy. Disclosures: No relevant conflicts of interest to declare.

Description: JAK2 V617F can be identified in the majority of cases of polycythemia vera (PV), and in 50% of cases in essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). JAK2 inhibitors including ruxolitinib (Jakavi, INCB018424), TG101348 and lestaurtinib (CEP-701) display clinical activity in clinical trials in PV, ET and IMF, and ruxolitinib has recently been approved for the treatment of primary and secondary myelofibrosis. In other malignancies it has been demonstrated that acquired resistance to kinase inhibitors is due to emergence of secondary resistance mutations in the target kinase. So far no JAK2 drug resistant mutations have been described in patients. To identify JAK2 point mutations mediating ruxolitinib resistance, we exposed JAK2 V617F expressing Ba/F3 cells to ruxolitinib. Surprisingly, sublines resistant to ruxolitinib at 1000nM, 2000nM and even 4000nM did not harbor point mutations neither in the kinase nor in the pseudokinase domain of JAK2. However, western blot analysis of sublines resistant to ruxolitinib revealed a 45-kDa JAK2 variant together with full length JAK2V617F protein in 87% of the cases. Sequencing of the short form in drug resistant clones revealed a novel JAK2 variant missing amino acids 76 to 820 resulting in the N-terminal FERM domain directly fused to the kinase domain of JAK2 (FERM-JAK2). FERM-JAK2 was highly resistant to the ATP-competitive JAK2 inhibitors ruxolitinib and TG101348. JAK2 exists in an either active or inactive state, which is largely regulated by phosphorylation of tandem tyrosines located within the activation loop. Substitution of phenylalanine at tyr1007, tyr1008 or both leads to complete inactivation of V617FJAK2 and WT-JAK2.In contrast, Y to F mutation of tyr1007, tyr1008 or both in FERM-JAK2 does not prevent activation of STAT5 and transformation of Ba/F3 cells, suggesting that FERM-JAK2 preferentially exist in an inactive state. This would impede drug binding and explain the resistant phenotype of FERM-JAK2. Phospho-deficient mutant studies further provided evidence that tyr866, tyr966, and tyr972 are crucial for the activation of FERM-JAK2, whereas in V617FJAK2 and WT-JAK2 tyr972, tyr1007 and tyr1008 were critical autophosphorylation sites. Ectopic expression of FERM-JAK2 in HEK293T, NIH3T3 and Ba/F3 cells showed activation of STAT5 and transformation of Ba/F3 cells to factor independence. Interestingly, co-immunoprecipitation studies revealed that FERM-JAK2 does not bind or activate the IL-3R in contrast to V617FJAK2. Ectopic expression of FERM-JAK2, in contrast to V617FJAK2, was able to activate STAT5 in a cytokine receptor deficient cell line. In vitro binding studies revealed that FERM-JAK2 directly binds and activates STAT5. Using flag- and myc-tagged FERM-JAK2 we show that the FERM domain is sufficient for an efficient dimerization and activation of FERM-JAK2 in the absence of an cytokine receptor. Transplantation of retrovirally transduced murine bone marrow with FERM-JAK2 compared to JAK2 V617F showed an accelerated phenotype with marked increases in WBC, HCT, RBC, HB, reticulocytes, pronounced splenomegaly and loss of body weight in mice. In contrast to V617FJAK2 expressing mice, FERM-JAK2 expressing mice displayed a lethal phenotype. Taken together, we could identify a novel JAK2 variant which alters the kinase domain structure, leads to direct, cytokine receptor independent STAT5 activation, and gives rise to an accelerated, PV-like disease in mice. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2423 Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) display limited activity in FLT3 mutant AML. Most patients experience primary or secondary TKI resistance. Only single cases of FLT3 kinase-dependent TKI resistance by secondary FLT3-ITD kinase domain mutations were reported. We therefore examined the mechanism of FLT3-ITD kinase-independent TKI resistance using an in vitro model. FLT3-ITD cell lines resistant to PKC412 or Sorafenib in 50% of the cases did not harbor secondary FLT3-ITD kinase domain mutations. However, 30% of these lines displayed a persistent phosphorylation of JAK2, IL-3βc and STAT5, while FLT3 phosphorylation was suppressed, indicating that these cell lines bypassed FLT3-ITD by activation of IL-3β by JAK2. Strikingly, TKI sensitive FLT3-ITD cells also displayed IL-3β phosphorylation, which in contrast to TKI resistant cells was JAK2 independent and was suppressed upon FLT3 TKI treatment. Expression of FLT3-ITD in IL-3β and JAK2 deficient g2A cells was sufficient for IL-3β phosphorylation. Co-IP experiments indicated a direct interaction of IL-3β and FLT3-ITD in TKI-sensitive and -resistant cells, which did not require interaction of IL-3β with IL-3a. This indicates that FLT3-ITD “physiologically” uses IL-3β as signaling intermediate. Mapping experiments showed that FLT3-ITD binding occurs in the membrane proximal region of IL-3β, independent of 8 tyrosines present in the cytoplasmic part. In search of the kinase bypassing FLT3-ITD in TKI-resistant cell lines, we identified an activating JAK1 mutation V658F in all TKI resistant FLT3-ITD cell lines displaying phosphorylation of JAK2, IL-3β and STAT5. JAK1 V658F is homologous to JAK2 V617F present in PV, phosphorylates IL-3β and STAT5, and confers TKI resistance in FLT3-ITD positive cells, which could be overcome by the JAK1/2 inhibitor Ruxolitinib. In summary, these data indicate that IL-3β not only mediates “physiological” FLT3-ITD dependent STAT5 activation, but also serves as a signaling module mediating JAK1/2-dependent TKI resistance. Thus, JAK inhibitors might represent an attractive therapeutic option in addition to FLT3 inhibitors in FLT3-ITD positive AML. Disclosures: Duyster: Novartis: Honoraria, Speakers Bureau. von Bubnoff:Novartis: Honoraria, Research Funding, Speakers Bureau.

Description: Background: Allogeneic hematopoietic cell transplantation is a potentially curative therapy for patients with hematological malignancies. However a fraction of patients will develop corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) which both cause a high mortality and impaired quality of life. Pre-clinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib by modification of T cells and dendritic cells. Methods: In this retrospective analysis, 19 stem cell transplant centers in Europe and the United States reported clinical outcome data from 95 patients who had received ruxolitinib as salvage-therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGvHD (1-10). The median follow-up times were 26.5 (3-106) for SR-aGVHD and 22.4 (3-135) weeks for SR-cGVHD-patients. Results: The ORR was 81.5% (44/54) in SR-aGVHD including 25 CRs (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). The median time to response was 1.5 (1-11) and 3 (1-25) weeks after initiation of ruxolitinib treatment in SR-aGVHD and SR-cGVHD, respectively. Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3%-90.7%,95% CI) and 97.4% (92.3%-100%,95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and CMV reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Relapse of the underlying malignancy occurred in 9.3% (5/54) and 2.4% (1/41) of the patients with SR-aGVHD or SR-cGVHD, respectively. Conclusion: Ruxolitinib constitutes a promising new treatment option for SR-aGVHD and SR-cGVHD. Its activity in SR-aGVHD and SR-cGVHD should be validated in a prospective trials in both, SR-aGvHD and cGvHD. Disclosures Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheid:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding.

Description: Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

Description: Introduction: Primary central nervous system lymphoma (PCNSL) relapses in up to 60% after conventional chemotherapy. The prognosis of refractory or recurrent PCNSL is very poor with a median survival of up to 5 months. Whole brain radiotherapy may improve survival up to 10 months, but is associated with significant neurotoxicity. High-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) have demonstrated high efficacy in the treatment of newly-diagnosed primary CNS lymphoma (PCNSL) in younger patients (pts.). To evaluate the efficacy of this approach, we initiated a prospective multicenter phase II study with HDT and ASCT for relapsed PCNSL. This trial is registered at ClinicalTrials.gov (NCT 00647049) Patients and Methods: Thirty eight pts.

Description: Introduction Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) were shown to be effective in clinical trials. However, complete remissions are rare, responses are short-lived, and the majority of patients display primary or secondary resistance to FLT3 inhibition. In FLT3 kinase inhibitor resistant, FLT3-ITD positive cell lines, we identified a direct interaction between FLT3-ITD and IL-3Rβc. We therefore sought to characterize this interaction and determine its' role in FLT3-ITD oncogenic signaling. Methods Drug sensitive BA/F3 FLT3-ITD cells and sublines resistant to FLT3 TKIs were subjected to analysis of activated IL-3Rβc - JAK1/2 - STAT signaling by PCR, Western-Blot (WB) and Immunoprecipitation (IP). Interaction studies were performed in vitro using IVT FLT3-ITD and GST IL-3Rβc, and in cell lines using γ2A cells transfected with human FLT3-ITD and human IL-3Rβc, human MOLM13 and MV4-11 AML cell lines, and primary AML patient samples by WB, IP and proximity ligation assay (PLA). Mapping experiments were performed in mouse embryonic fibroblasts (MEF) transduced with FLT3-ITD and flag-IL-3Rβc full-length or cytoplasmic Y to F mutant constructs. Knockdown-experiments were performed in BA/F3 cells with stably transfected FLT3-ITD and inducable IL-3Rβc shRNA. Results In TKI resistant FLT3-ITD positive cell-lines that did not harbor secondary FLT3 mutations inducing TKI resistance, we observed phosphorylation of IL-3Rβc in 5/16 resistant lines (30%) in the presence of FLT3 TKI. In these cells, IL-3Rβc phosphorylation was mediated by an activating JAK1 V658F mutation that bypasses FLT3-ITD dependent IL-3Rβc phosphorylation. Thus, IL-3Rβc in FLT3-ITD expressing cells mediates JAK1/2-dependent TKI resistance. Of note in inhibitor sensitive cells, IL-3Rβc interacted with and was phosphorylated by FLT3-ITD in a JAK1/2 independent manner, suggesting that IL-3Rβc participates in FLT3-ITD dependent oncogeneic signaling. Indeed, in IL-3Rβc and JAK2 deficient γ2A cells, expression of human FLT3-ITD was sufficient to induce interaction with and phosphorylation of human IL-3Rβc. In human FLT3-ITD-positive AML cell lines MOLM13 and MV4-11, IL-3Rβc phosphorylation occurred in a FLT3-ITD dependent fashion and IL-3Rβc interacted with FLT3-ITD. Proximity ligation assay (PLA) experiments detected FTL3-ITD and IL-3Rβc in close proximity suggesting a direct interaction of both proteins in MOLM13 and MV4-11 cells, as well as in primary cells from FLT3-ITD positive AML patients. Interaction studies revealed that binding to FLT3-ITD occurred independent of IL-3Rβc cytoplasmic tyrosines, and phosphorylation of IL-3Rβc by FLT3-ITD did not require the presence of IL-3Rαc. Preliminary results from knock down experiments in FLT3-ITD expressing BA/F3 cells indicated that knock-down of IL-3Rβc renders cells more susceptible to FLT3 TKI inhibition. Conclusion These data suggest an entirely novel model of FLT3-ITD "physiologically" employing IL-3Rβc as signaling intermediate, and IL-3Rβc serving as a signaling module mediating JAK1/2-dependent TKI resistance. These findings point toward the significance of IL-3Rβc for FLT3-ITD dependent transformation and treatment resistance, supporting the relevance of IL-3Rβc as a possible treatment target in FLT3-ITD positive AML. Disclosures von Bubnoff: Novartis: Research Funding; BMS: Speakers Bureau.