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Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final “common path.” Clin Cancer Res; 23(16); 4805–16. ©2017 AACR.

S1 BAX/BAK localization for the test cohort S2 clinical characterization of patient cohorts S3 GISTIC analysis of the test cohort S4 BAX/BAK localization for the validation cohort S5 Multivariate analysis of variance for human AML survival probablity

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

Tyrosinkinase-Inhibitoren stellen einen wichtigen Beitrag zur Weiterentwicklung und Verbesserung der Therapie von hamatologischen Neoplasien dar. Zahlreiche Inhibitoren befinden sich bereits in fortgeschrittener klinischer Testung. Wahrend bei einem Teil der Erkrankungen (CML) die Inhibitor-Therapie bereits als Standard etabliert ist, befinden sich die “small molecules” bei Akuter Myeloischer Leukamie und chronisch-myeloproliferativen Syndromen noch in der Etablierungsphase. Die Entwicklung von neuen zielgerichteten Substanzen zahlt zu den grosen praklinischen und klinischen Herausforderungen der nachsten Jahre.

SummaryTreatment of relapsed or refractory autoimmune mediated haemolytic syndromes, such as autoimmune haemolytic anaemia (AIHA) and thrombotic thrombocytopenic purpura (TTP), represents a therapeutic challenge. Here we report on our experience with the monoclonal anti-CD20 antibody rituximab (R) compared to standard treatment in these diseases. Patients with non-familialTTP orAIHA and no underlying malignancy were included in our analysis. Safety and efficacy of R-treatment were compared to results obtained in standard treatment approaches. Altogether, 27 patients were analyzed, comprising 15 patients withTTP and 12 patients with AIHA. The patients’ average age at the time of diagnosis was 54 years. Eleven patients received antibody treatment (8 TTP, 3 AIHA). No acute or late WHO grade III/IV toxicity associated with rituximab was noted. With standard therapy, the overall response rate (ORR) was 66.7% for AIHA and 65.8% for TTP, respectively. For the R-containing regimens the ORR was 100%. In patients with TTP, median progression free survival (PFS) with R-treatment was 3.8 years, as compared to 0.1 years in the standard-treatment group. In patients with AIHA median PFS was not reached for R-containing treatment; all patients are in sustained remissions with a median follow up of 12.5 months. In the absence of prospective trials, our data underline the safety and efficacy of rituximab in relapsed and refractory autoimmune anaemias with favourable response rates and promising long-term progressionfree survival. Therefore, prospective clinical trials evaluating rituximab as salvage- and first-line-therapy are clearly warranted.

Activating mutations of FLT3 are found in approximately one-third of acute myeloid leukemia (AML)-cases and are considered to represent an attractive therapeutic target. In this study, we report that the hydroxystyryl-acrylonitrile compound LS104 inhibits proliferation and induces potent cytotoxic effects in FLT3 expressing leukemic cells in vitro. Immunoblot and phosphoprotein-FACS analysis demonstrated inhibiton of phosphorylation of FLT3-ITD and of its downstream targets. In pharmacokinetic studies, a rapid and dose dependent cellular uptake of LS104 lasting up to 11h could be demonstrated. Combination of LS104 with chemotherapeutic agents markedly enhanced cytotoxic effects. Recently, a phase I clinical trial investigating LS104 in refractory/relapsed hematologic malignancies has been initiated.

This study aimed to investigate the outcome of patients with advanced gastrointestinal stromal tumors (GISTs) exhibiting focal disease progression during imatinib therapy, treated by surgical resection and imatinib continuation.A consecutive series of 38 patients with metastatic GISTs who underwent treatment with imatinib at our centers during a defined period of time was evaluated. Patients were evaluated for demographics including tumor-related features, initial response, disease recurrence, and salvage treatment modalities, and were classified as having either focal or generalized progression upon presentation prior to salvage therapy.After a median follow-up of 31.8 months, 25 of the 38 (65.8%) patients had progressed. Nine (36%) patients were classified as having focal and 16 (64%) as having generalized progression. Salvage therapies were: surgical resection and imatinib dose escalation in patients exhibiting focal progression and imatinib dose escalation alone in the majority of patients exhibiting generalized progression. Focal progression was associated with prolonged progression-free survival (PFS) and overall survival (OS) after salvage therapy as compared with generalized progression (median PFS and OS, 11.3 months and not attained, versus 2.5 and 22.8 months, respectively). Six-month PFS was 89% and 39% in patients exhibiting focal and generalized progression, respectively. KIT mutation analysis of controlled and progressive lesions was performed in 4 patients with focal progression. Secondary KIT mutations affected progressive lesions, whereas nonprogressive lesions harbored the original mutations only.Patients with advanced GIST exhibiting focal disease progression during imatinib therapy may benefit from surgical resection and imatinib continuation. Imatinib resistance seems to be partial in these patients.

Internal tandem duplication (ITD) mutations of FLT3 are present in leukemic blasts of approximately 30% of AML patients. ITD-mutations of FLT3 confer a worse prognosis and decreased overall survival. Therefore, FLT3-tyrosine kinase is considered an attractive drug target in AML and several FLT3-tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials (CEP701, MLN518, Sorafenib, PKC412). However, using these drugs as monotherapy, against the setting of remarkable efficacy has emerged the problem of short duration of remission indicating rapid development of secondary resistance. In addition, up to 30% of patients may show primary resistance to currently available FLT3-TKIs. Therefore, to overcome these limitations further clinical development requires novel approaches (e.g. combination with chemotherapy) and development of more efficacious FLT3-TKI. Here, we investigated two novel TKIs (3,4-Diarylmaleimides, compounds 53 and 80) previously described as angiogenesis inhibitors (Peifer, J. Med. Chem. 2006), to determine their mechanism of action and efficacy in FLT3-ITD transfected 32D cells as well as in primary ITD-positive AML blasts. Western Blotting experiments of 32D-FLT3-ITD cells confirmed dose dependent inhibition/dephosphorylation of downstream targets as STAT5 (pTyr694/699), ERK (pThr202/pTyr204) and AKT (pSer473) upon incubation with either compound using nanomolar concentrations. Both compounds (cpd.) showed induction of apoptosis with an IC50 of 350nM (cpd. 53) and 800nM (cpd. 80), respectively, as detected by sub G1-fraction in cell cycle analysis. Using colony assays we defined a possible therapeutic window of these inhibitors: Bone-marrow cells of healthy donors were incubated with increasing concentrations of both inhibitors revealing no significant inhibition of colony formation up to a dose of 5 μM. As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both cpds. in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Within a range of 1–50nM DNR and 0.3–10μM Ara-C, both cpds. showed additive effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays. Primary AML blasts harboring the FLT3-ITD length mutation were also investigated to determine the efficacy in patient material. These experiments revealed apoptosis rates of 10–30% upon incubation with 3,4-Diarylmaleimides for 72 hours. Cellular up-take of cpd. 53 in primary AML blasts could be demonstrated within 5 minutes of incubation using FACS analysis. Increased fluorescence (200–400% of baseline values) could be detected for more than 24 hours suggesting stable intracellular concentration of the inhibitor in AML blasts within this time period. To evaluate signaling targets of 3,4-Diarylmaleimides in detail, we applied a kinomics peptide chip containing a set of 960 kinase substrates (human sequences only) in triplicate on a slide. Phosphorylation of peptide substrates was detected by autoradiography. Results of evaluation will be presented. In conclusion, 3,4-Diarylmaleimides show efficacy in FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Currently, experiments evaluating toxicity in murine models are planned to explore a possible clinical application.

The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase inhibitor. Ba/F3 cells stably transfected with EpoR and either the V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT) were treated with LS104. Apoptosis assays as well as immunoblotting of JAK2 and of downstream signalling pathways were performed. The effects of LS104 on kinase activity were determined in an in vitro JAK2 kinase assay. A combination of LS104 with JAK-inhibitor I, which acts via the ATP-binding site, was tested in apoptosis assays. Finally, growth of endogenous erythroid colonies (EECs) obtained from patients with V617F-positive MPD was tested upon addition of LS104. LS104 selectively and dose dependently induced apoptosis in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT control cells. By immunoblotting we found inhibition of JAK2 autophosphorylation and of downstream targets as STAT5, AKT and ERK upon treatment with LS104. Activation of these targets by JAK2 was confirmed in experiments using JAK2 siRNA. The IC50 for JAK2 in an in vitro kinase assay using LS104 was

Fms-like tyrosine kinase 3 (FLT3), a member of the class III tyrosine kinase receptor family, is expressed in up to 90% of acute myeloid leukemia (AML). Activating mutations like internal tandem duplication (ITD) of the juxtamembrane domain and kinase domain point mutations are found in approximately 35% of AML-cases and are considered to represent an attractive therapeutic target. In this study, we report that the novel hydroxystyryl-acrylonitrile compound LS104 induces potent cytotoxic effects in FLT3 ITD-positive leukemic cells. As a cellular model to investigate FLT3-ITD specific effects we used 32D myeloid cells stably transfected with FLT3-ITD and wt-FLT3, respectively. In MTS assays, pronounced inhibition of cell growth was seen at nanomolar concentration (IC50=50nM) which could be partially rescued by addition of IL3. LS104 at a concentration ranging from 3–10μM readily induced apoptosis as evaluated by cell cycle analysis, annexin-V assays and PARP cleavage. Throughout the experiments, FLT3-ITD expressing cells showed significantly higher sensitivity towards LS104 than cells expressing wt-FLT3. Similar results were observed evaluating the cytotoxic effects of LS104 in the human myeloid-leukemia derived FLT3-ITD harbouring MV4;11 and the wt-FLT3 expressing lymphoid-leukemia derived RS4;11 cell line. Immunoblot analysis demonstrated that LS104 inhibits tyrosine phosphorylation of FLT3-ITD and of its downstream target STAT5. This points out that FLT3-ITD is a molecular target of LS104. Interestingly, efficacy of LS104 to induce apoptosis was significantly reduced in 32D cells transfected with a FLT3-ITD isoform (N676K) previously reported to be associated with clinical resistance of FLT3-ITD to the kinase inhibitor PKC412 (Heidel et al., ASH 2004). This result strongly suggests that the mechanism of action of LS104 is indeed inhibition of FLT3 kinase activity rather than inhibition of other kinases targeted by LS104. As clinical development of FLT3 kinase inhibitors in AML likely will be in combination with chemotherapy, we evaluated the in vitro effects of combining LS104 with the cytostatic drugs cytosine arabinosid (Ara-C) and daunorubicin (DNR). In 32D FLT3-ITD cells, cell cycle analysis showed strong synergy in induction of apoptosis upon incubation with LS104 (1–3μM) plus Ara-C (1μM) and DNR (10nM), respectively. Using primary blasts from AML patients, we further evaluated cytotoxicity of LS104 at various concentrations and in combination with Ara-C and DNR, respectively. LS104 as a single agent was shown to induce apoptosis in primary AML blasts and combination of LS104 with cytostatic drugs resulted in additive and synergistic effects in leukemic blasts from 4 out of 6 patients investigated. Western-blotting of primary AML blasts revealed inhibition of FLT3-ITD and STAT5 tyrosine phosphorylation by LS104 in a dose-dependent way (3–50μM). Interestingly, LS104 showed intrinsic fluorescence activity. Using FACS analysis and fluorescence microscopy we could demonstrate a rapid and dose-dependent cellular uptake of LS104 in leukemic cell lines and in primary blasts. After removal of LS104 from medium, enhanced fluorescence corresponding to intracellular LS104 was detectable up to 24 hours. This property could be used to monitor drug uptake of primary AML blasts in vivo. In conclusion, our data provide a preclinical framework for clinical trials of LS104 in FLT3-ITD positive leukemia.

The FLT3 receptor tyrosine kinase is expressed in 70-90% of cases of AML. Up to 35% of patients with AML show mutations in the JM-region or kinase domain of FLT3. These lead to autophosphorylation promoting ligand-independent cell proliferation and inhibition of apoptosis. Treatment with FLT3 tyrosine kinase inhibitors (TKI) is a promising tool in therapy of AML. Preliminary results investigating the FLT3-TKI PKC412 in patients with relapsed/refractory AML revealed that 11/15 patients (73%) with mutated FLT3 and 16/46 patients (35%) with WT FLT3 showed a >50% blast response in peripheral blood (Estey E et al. Blood.2003; 102:919a). Despite its remarkable efficacy in reducing the leukemic clone, remissions upon single agent therapy using PKC412 tend to be short and secondary resistance occurs rather rapidly. So far, very little is known on the mechanisms of resistance to therapy using FLT3-TKIs. Therefore, we sought to characterize the molecular mechanisms involved. Overall PB and BM samples from 5 patients undergoing single agent therapy with PKC412 were analyzed. Three patients revealed reactivation of FLT3-kinase at relapse. Unchanged protein levels of Flt3 as detected by Western blotting before therapy and at relapse showed that amplification of FLT3 does not appear to play a role in development of secondary resistance. PKC412 plasma levels, as measured by HPLC, were similar in patients with and without reactivation of the kinase. Therefore, we examined the bioactivity of plasma obtained at relapse to inhibit phosphorylation of FLT3 in MV4-11 cells harboring FLT3-ITD. In two patients, plasma inhibited FLT3-tyrosine phosphorylation at the time of remission as well as at relapse, while in one patient, plasma was not able to inhibit FLT3-phosphorylation. Therefore, in this patient, an inhibitory activity in serum appeared to be operating. A potential candidate for this inhibitory activity is alpha1-glycoprotein (AGP) that was shown previously to mediate resistance to STI571 treatment. Our experiments showed that AGP is indeed able to inhibit bioactivity of PKC412 in-vitro. However, AGP-levels monitored in PB samples from patients included in this analysis did not show elevated plasma levels at relapse. Ex-vivo treatment of primary blasts with PKC412 at relapse showed that in one patient, blasts were still sensitive to PKC412. In two other patients, blasts showed resistance using a wide dose range of PKC412. Therefore, in these patients, cDNA sequencing of FLT3 was performed. In one patient, we identified a point mutation leading to an aminoacid exchange within the N-lobe of the FLT3-kinase domain at relapse. This mutation was not present at start of PKC412 therapy. Experiments analyzing the functional role of the novel mutation in the hematopoietic cell line 32D are in progress and will be presented. In conclusion, we have preliminarily identified examples of kinase-dependent and independent mechanisms operating in clinical resistance to PKC412.

The receptor tyrosine kinases (RTKs), epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 1-3 (VEGFR1-3), are frequently expressed in gastric cancer and are putative therapeutic targets in this disease. We have investigated the anti-proliferative and chemosensitizing properties of the multitargeted small-molecule RTK inhibitors sunitinib and vandetanib in a panel of 4 human gastric and esophageal cancer cell lines. In the 1st instance, the expression of potential targets of these small-molecule inhibitors was examined by reverse transcriptase-polymerase chain reaction, western blotting, and flow cytometry. EGFR mRNA and protein was detected in all cases, with VEGFR2 expression noted in all but 1 line. Both EGF and VEGF were shown to stimulate tumor cell growth, and both sunitinib and vandetanib were found to be associated with significant dose-dependent inhibition of proliferation and an enhancement of apoptosis, as determined by MTT and propidium iodide/Annexin V labeling assays, respectively. The addition of sunitinib to VEGF-stimulated NCI-N87 cells was associated with a reduction in MAPK phosphorylation (pMAPK) but not Akt phosphorylation (pAkt), whereas the addition of vandetanib was associated with reductions in both VEGF- and EGF-mediated VEGFR2 phosphorylation, pMAPK and pAkt. Co-administration of sunitinib significantly enhanced the sensitivity of MKN-45 cells to cisplatin and irinotecan. In addition, vandetanib synergistically enhanced the sunitinib-associated inhibition of gastric cancer cell growth. In conclusion, these preliminary data confirm the importance of EGFR and VEGFR signaling in gastric cancer and suggest that the simultaneous inhibition of RTK-pathways through sunitinib and vandetanib may provide therapeutic benefit in this disease.

Comorbidities defined by the Charlson comorbidity index (CCI) and body mass index (BMI) are significantly associated with outcome in patients who receive continuous treatment with tyrosine kinase inhibitors. We evaluated the impact of CCI and BMI on responses, drug-related toxicities, and outcome in a cohort of 402 patients with myelofibrosis (MF) treated with ruxolitinib in 23 European Hematology Centers. Comorbidities were evaluable in all 402 patients. A higher (≥ 3) CCI did not correlate with a lower spleen reduction at any time (p = 0.68) or symptoms' response (p = 0.11), but influenced the onset of anemia during the first 3months of treatment and later (p = 0.02 and p = 0.03, respectively) in patients without anemia baseline. BMI was evaluable in 380 patients and did not correlate with differences in spleen and symptoms response (p = 0.57 and p = 0.49, respectively). A higher CCI and a lower BMI correlated also with a reduced overall survival (p

Ruxolitinib is a JAK1/2 inhibitor that may control myelofibrosis (MF)-related splenomegaly and symptoms and can be prescribed regardless of age. While aging is known to correlate with worse prognosis, no specific analysis is available to confirm that ruxolitinib is suitable for use in older populations. A clinical database was created in 23 European Haematology Centres and retrospective data on 291 MF patients treated with ruxolitinib when aged ≥65 years were analysed in order to assess the impact of age and molecular genotype on responses, toxicities and survival. Additional mutations were evaluated by a next generation sequencing (NGS) approach in 69 patients with available peripheral blood samples at the start of ruxolitinib treatment. Compared to older (age 65-74 years) patients, elderly (≥75 years) showed comparable responses to ruxolitinib, but higher rates of drug-induced anaemia and thrombocytopenia and worse survival. Nonetheless, the ruxolitinib discontinuation rate was comparable in the two age groups. Number and types of molecular abnormalities were comparable across age groups. However, the presence of high molecular risk (HMR) mutations significantly affected survival, counterbalancing the effect of aging. Indeed, elderly patients with