ALBERT

Description: Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations. Disclosures No relevant conflicts of interest to declare.

Description: The most common chromosomal translocation in acute promyelocytic leukemia (APL), t15;17(q22;q21), creates PMLRAR andRARPML fusion genes. We previously developed a mouse model of APL by expressing PMLRAR in murine myeloid cells. In order to examine the mechanisms by which PMLRAR can initiate leukemia, we have now generated transgenic mice expressingPMLRARm4 and RARm4, proteins that are unable to activate transcription in response to retinoic acid.PMLRARm4 transgenic mice developed myeloid leukemia, demonstrating that transcriptional activation by PMLRAR is not required for leukemic transformation. The characteristics of the leukemias arising in the PMLRARm4 transgenic mice varied from those previously observed in our PMLRAR transgenic mice, indicating that ligand responsiveness may influence the phenotype of the leukemic cells. The leukemias that arose in PMLRARm4transgenic mice did not differentiate in response to retinoic acid therapy. This result supports the hypothesis that a major therapeutic effect of retinoic acid is mediated directly through thePMLRAR protein. However, a variable effect on survival suggested that this agent may be of some benefit in APL even when leukemic cells are resistant to its differentiative effects. Transgenic mice expressing high levels of RARm4 have not developed leukemia, providing evidence that the PML domain ofPMLRAR plays a specific and critical role in the pathogenesis of APL.

Description: There is conflicting information about the influence of body mass index (BMI) on the pharmacokinetics, toxicity, and outcome of chemotherapy. We compared pharmacokinetics, outcome, and toxicity data across 4 BMI groups (underweight, BMI ≤ 10th percentile; normal; at risk of overweight, BMI ≥ 85th and 〈 95th percentile; overweight, BMI ≥ 95th percentile) in 621 children with acute lymphoblastic leukemia (ALL) treated on 4 consecutive St Jude Total Therapy studies. Chemotherapy doses were not adjusted to ideal BMI. Estimates of overall survival (86.1% ± 3.4%, 86.0% ± 1.7%, 85.9% ± 4.3%, and 78.2% ± 5.5%, respectively; P = .533), event-free survival (76.2% ± 4.2%, 78.7% ± 2.1%, 73.4% ± 5.5%, and 72.7% ± 5.9%, respectively; P = .722), and cumulative incidence of relapse (16.0% ± 3.7%, 14.4% ± 1.8%, 20.6% ± 5.1%, and 16.7% ± 5.1%, respectively; P = .862) did not differ across the 4 groups. In addition, the intracellular levels of thioguanine nucleotides and methotrexate polyglutamates did not differ between the 4 BMI groups (P = .73 and P = .74, respectively). The 4 groups also did not differ in the overall incidence of grade 3 or 4 toxicity during the induction or postinduction periods. Further, the systemic clearance of methotrexate, teniposide, etoposide, and cytarabine did not differ with BMI (P 〉 .3). We conclude that BMI does not affect the outcome or toxicity of chemotherapy in this patient population with ALL.

Description: Abstract 601 Background. DCC-2036 is a novel and potent tyrosine kinase inhibitor (TKI) which binds to a novel region called the switch pocket, thereby preventing BCR-ABL from adopting a conformationally active state. Efficacy against multiple imatinib-resistant BCR-ABL mutants has been demonstrated both in vitro and in vivo (Chan et al., Cancer Cell 2011;19:556). Importantly, DCC-2036 retains full potency against the T315I mutant in preclinical efficacy studies. Methods. This study was designed to find the maximal tolerated dose (MTD) of DCC-2036 when administered daily as a single-agent on a 28-day cycle. Eligible patients included adults with Ph+ CML/ALL who were refractory/intolerant to ≥2 TKI's or were T315I positive. Initially DCC-2036 capsules were administered orally once daily (QD) at increasing dose levels. Only 1 patient was enrolled in each of the lowest dose cohorts of 57mg QD and 114 mg QD. For higher doses, 3– 6 patients were enrolled into each ascending dose cohort with standard dose limiting toxicity (DLT) rules evaluating safety in cycle 1 to determine dose escalation. A transition from unformulated capsules (C) to formulated tablets (T) occurred after the 1200 mg QD dose level. Paired blood samples were obtained for PK and PD assessments. Results. 30 patients (16 males, 14 females; median age 59, range 31 – 80) with CML including 19 in Chronic (CP); 8 in Accelerated (AP) and 3 in Blast (BP) Phase were enrolled. Enrolled patients had received 1–6 prior CML treatments, and 11 patients had the T315I mutation. To date, a total of 212.5 (median 5.6; range 0.2 – 23.4) 28-day cycles were administered over 10 dose levels either as C (7 dose levels) or T (3 dose levels). The 7 C dose levels were studied first and included 57 mg QD through 1200 mg QD. Following transition to T, evaluation continued with 100 mg QD, 100 mg twice daily (BID), and 200 mg BID. Two reversible DLTs (Grade 3 peripheral neuropathy and Grade 4 lower extremity weakness) occurred during the initial treatment cycle at the 200 mg T BID dose level. Evaluation of 6 patients at the 150 mg T BID dose level determined that dose to be the MTD. Preliminary safety data show that other Grade (Gr) 3/4 adverse events (AEs) were Gr 3 slurred speech and Gr 3 eruptive nevi. Gr 1/2 AEs included dry mouth, constipation, diarrhea, paresthesias, and retinal vein occlusion. There was 1 case of Gr 2 pancreatitis that recurred on rechallenge in a patient with previous pancreatitis with nilotinib. Preliminary responses include one major molecular response in a CP patient with T315I mutation who started on capsules and transitioned to 100 mg T QD. There was one complete cytogenetic response in a CP patient at 100 mg T BID, and one partial cytogenetic response in a CP patient who started on capsules and transitioned to 100 mg T BID. One patient with AP CML and T315I mutation had a complete hematologic response at 450 mg C QD. Another patient with AP CML had a partial hematologic response after receiving 200 mg BID for 1 cycle and then downdosing to 100 mg T BID. Four out of 8 patients receiving 100 mg tablets and evaluable for efficacy (completed 3 cycles of treatment) had responses. PK results indicate dose-related, nonlinear increases in both peak plasma concentration (Cmax) and exposure (AUC). PD results reveal both acute and steady state post-treatment reductions in phospho-protein levels on Days 1 and 8. Marked reductions in pSTAT5 and pCRKL have been observed in subjects with both CP and AP and appear to be required for clinical response. Conclusion. The MTD of DCC-2036 tablets is 150 mg BID. Preliminary results suggest that DCC-2036 is well tolerated and has anti-leukemia activity in subjects with refractory CML and T315I positive disease. PD results are consistent with inhibition of BCR-ABL signaling in this first-in-man study of a switch pocket tyrosine kinase inhibitor. Disclosures: Cortes: Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Chemgenex: Consultancy, Research Funding; Deciphera Pharmaceuticals: Research Funding. Bixby:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; GlaxoSmithKline: Speakers Bureau. Rafferty:Deciphera Pharmaceuticals: Employment. Berger:Deciphera Pharmaceuticals: Employment. Wise:Deciphera Pharmaceuticals LLC: Employment. Rutkoski:Deciphera Pharmaceuticals: Employment. Smith:Deciphera Pharmaceuticals: Employment. Van Etten:Deciphera Pharmaceuticals: Consultancy, Research Funding.

Description: Introduction: Currently, patients with refractory Hodgkin lymphoma (HL) or those who relapse multiple times have an extremely poor prognosis. Even patients who do not relapse often experience late toxicities, including secondary cancer, heart failure, pulmonary dysfunction, and infertility. Therefore, new agents and novel therapeutic approaches are urgently needed. Anti-apoptotic proteins such as Bcl-2 and Bcl-xl have been found to be associated with the growth and survival of Hodgkin Reed–Sternberg cells and thus carry the potential to be effective targets for therapeutics. In this study we report the in vitro cytotoxicity, biological correlative findings and drug combination analysis of the novel BH3 mimetic ABT-737 [Abbott laboratories (Abbott Park, IL)] against HL cells. Materials and Methods: HL cell lines (KMH2 and HDLM2) were cultured in the presence of increasing concentrations of ABT-737 or its enantiomer control. Normal bone marrow stromal cells were used as controls for non-specific cytotoxicity. Cell growth inhibition was measured by Alamar blue assay and the induction of apoptosis was demonstrated by an annexin specific staining technique. Time and drug concentration dependent changes in proteins involved in cell survival and apoptosis were investigated by Western blot analysis. The ability of ABT-737 to influence the anti-lymphoma activity of a panel of twenty distinct chemotherapeutic agents was evaluated by drug combination and cell growth inhibition studies. Combination indices (CI) were calculated to identify therapeutic enhancement of different agents in the presence of Bcl-2 inhibition by ABT-737. Results: ABT-737 showed significant dose-dependent cytotoxicity and apoptotic activity against HL cells with an approximate IC50 of 1mM with maximum cell death occurring at about 5mM. At the molecular level, increased levels of cleaved caspase3 and PARP, as well as annexin positivity, were noticeable within three hours of treatment with the agent. Drug combination studies have shown the ability of Bcl-2 inhibition to synergize with novel therapeutic agents that target histone deacetylase function (Apicidin, CI 0.35), Hsp90 stability (17-AAG, CI 0.3) and the activity of specific receptor tyrosine kinases (Sorafenib, CI 0.7 and Sunitinib CI 0.3 for HDLM2 and CI 0.9 for KMH2). Among conventional anti-neoplastic agents, ABT-737 showed significant synergistic activity with irinotecan and oxaliplatin (CI 0.6). Importantly, treatment with ABT-737 decreased the expression of the critical HL cell growth promoter, NF- B as determined by band densities on Western blot analysis: a two fold decrease in KMH2 and a five fold decrease in HDLM2 cells. Specific target modulation was demonstrated by changes in key apoptosis and cell survival regulators such as Bcl-XS/L, Bcl-X, p53 and survivin by two to 10 fold decreases in Western blots. Discussion: Data presented in this study support the hypothesis that Bcl-2 family of proteins can be an effective target for therapeutics in HL. We have shown that the BH3 mimetic ABT-737 induces apoptosis in these cells, characterized by the modulation of key components of cell growth and survival pathways. In addition, we have identified distinct classes of anti-lymphoma and anti-neoplastic agents whose activities are enhanced by concurrent inhibition of Bcl-2. These findings provide the rationale for further evaluation of ABT-737 and the subsequent clinical development of a targeted anti-Bcl-2 therapy for refractory Hodgkin lymphoma.

Description: Abstract 3163 The KCl co-transporter (KCC) family of proteins catalyzes the electroneutral, coupled movement of K+ and Cl− ions across the plasma membrane, thereby mediating transepithelial ion transport and regulating cell volume. These proteins play an important role in disease states such as cancer, numerous neurological conditions as well as sickle cell disease (SCD). KCC activity is increased in sickle red blood cells and contributes to their dehydration, which potentiates sickling. The mechanisms of increased KCC activity and its abnormal regulation are not understood. Of the four mammalian KCC isoforms, KCC 1, 3 and 4 are expressed in erythroid cells (Crable et al. Exp Hematol. 2005; 33:624). Hiki et. al. showed that the angiogenic factor vascular endothelial growth factor (VEGF) increased KCC 3a expression in HUVEC cells (J.B.C. 274, 10661–10667, 1999). As levels of VEGF and related family member, placenta growth factor (PlGF) are elevated in sickle cell patients, we hypothesized that VEGF and PlGF may influence KCC expression in erythroid cells. RT-PCR revealed that erythroid K562 cells expressed the VEGF receptor-1 (VEGF-R1, or Flt-1) but not VEGF receptor-2, (VEGF-R2 or Flk-1). Additionally, flow cytometric analysis of WT C57Bl6 mouse bone marrow showed the presence of the Flt-1 receptor, but not Flk-1 or Flk-3 in erythroid progenitors and expression decreased with maturation. VEGF treatment (50 ng/ml) of K562 cells increased KCC 1, 3a, 3b and 4 mRNA levels; PlGF treatment increased KCC 1, 3a and 4 mRNA levels but not KCC 3b. The VEGF receptor inhibitor, SU5416, ablated the effect of VEGF. VEGF-stimulated KCC 4 expression was blocked by pharmacological inhibitors that implicated PI3 kinase, p38 MAP kinase, mTOR, JNK kinase and the transcription factor hypoxia inducible factor-1α (HIF-1α), as with other VEGF effects. Analysis of the KCC 4 promoter showed that the −875 and −90 bp promoter luciferase constructs exhibited similar levels of activity as the −1200 bp promoter construct, when compared to the promoterless reporter plasmid. Deleted constructs corresponding to −65 bp from transcription start site showed ∼90% reduced promoter activity. In silico analysis of the −90 bp region of the KCC 4 promoter showed potential binding sites for transcription factor SP-1 and HIF-1α. Binding sites for transcription factor SP-1 at positions −35 to −44 bp and −56 to −64 bp were shown to be active by site directed mutagenesis. Mutation of the HIF-1α binding site at −73 to −76 bp significantly inhibited promoter activity, whereas mutation of the HIF-1α binding site at position −21 bp to −18 bp did not have any effect on activity. Similar analysis of the KCC 3a promoter indicate potential binding sites for SP-1 at positions −8 to −4 bp and a HIF-1α binding site at position −23 to −20 bp, and the KCC 3b promoter has binding sites for HIF-1α at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at position −13 to −10 bp. Luciferase assays with KCC 3b promoter constructs indicated that the −190 bp promoter region containing HIF-1α sites at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at −13 to −10 bp contained minimal promoter required for transcription activity. Mutations within both HIF-1α binding sites attenuated promoter activity indicating a role for HIF-1α in regulating KCC 3b activity, as well. EMSA and ChIP assays with the KCC 4 promoter demonstrated that VEGF treatment of K562 cells increased HIF-1α binding to the HIF-1α sites, which was abrogated by mutating these sites. Similar results were obtained for the KCC 3a and 3b promoters.These results suggest that activation of VEGF-R1 by VEGF, and presumably its other ligand, PlGF, leads to non-hypoxic activation of HIF-1α and SP-1-mediated up-regulation of KCC3a, 3b and 4 expressions in erythroid K562 cells via its canonical signaling pathways. Variation in KCC gene expression and its modulation by cytokines and growth factors may be a source of inter-individual variation in SS RBC volume regulation and thus of phenotypic variability of SCD. Identifying the factors that modulate transcriptional control of KCC expression is important to understanding volume regulation in reticulocytes and its dysregulation in SS RBC. Disclosures: No relevant conflicts of interest to declare.

Description: OBJECTIVES: Activating RAS mutations and over-expression of BCL-2 are prognostic features of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) transformation. Using NRASD12 and BCL-2, we created two distinct transplantable in vivo models of MDS and AML. Expression of hBCL-2 in a primitive compartment by MMTV-LTR results in a disease resembling human MDS with bone marrow blasts of 15% with increased apoptosis assayed by TUNEL on liver sections, whilst the myeloid MRP8 promoter induces a disease with characteristics of human AML with marrow blasts of up to 90% with liver apoptosis patterns similar to wild type. In MDS mice RAS and BCL-2 do not co-localize in the mitochondria, but localize to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localize in the mitochondria, where BCL-2 is normally found; consistent with its anti-apoptotic properties. We next examine the mouse hematopoietic FDCP-1 in vitro model with stable exogenous expression of NRASD12, hBCL-2 or NRASD12 and hBCL-2. Furthermore, we report the in vivo effects of the BH-3 mimetic inhibitor ABT-737. RESULTS: FDCP-1 lines demonstrate that whilst hBCL-2 alone prevents staurosporine-induced mitochondrial-dependent (caspase-9-mediated intrinsic) apoptosis, both NRAS-D12 and NRASD12/hBCL-2 cell lines were pro-apoptotic. No significant difference was observed in cell-death receptor (caspase-8-mediated extrinsic) apoptosis between the cell lines following pharmacological induction with Fas-ligand or TRAIL. Annexin-V/Propidium Iodide flow cytometry and caspase activity assays on hematopoietic primary cells from the mouse models recapitulated the findings; namely the MDS-model demonstrates increased caspase-9-mediated apoptosis (within the Lin−/Sca-1+/KIT+ (LSK) sub-population), whilst the AML-model illustrate anti-apoptotic activity. This is concordant with the signaling profile where phosphorylated AKT is reduced in the RAS-mediated MDS mice and active-AKT is increased in the BCL-2 mediated AML disease. Expanded leukemic stem cell LSK populations had increased hBCL-2 expression in the RAS-GTP complex in both MDS/AML diseases. Thus we suggest that this complex can be a specific target for therapy. When hBCL-2 is switched off with doxycycline in the conditional MDS mice, only partial reversal of the phenotype was observed as RAS recruits endogenous mouse BCL-2 to remain active; thus demonstrating the role of the complex in the disease. In order to target both human and mouse BCL-2 the efficacy of in vivo treatment with the specific BH-3 mimetic inhibitor ABT-737 was determined. We show that the treatment significantly reduced the progression of the disease, increased the peripheral blood platelet counts, decreased the bone marrow blast and cleared the tissue invasion in the MDS mice or reduced tissue infiltration of the AML. In vivo imaging by single-photon emission computed tomography (SPECT) using 99mTc-labelled Annexin-V shows that ABT-737 induces apoptosis of the blast cells that infiltrate the liver and the spleen of the treated mice, which was confirmed by TUNEL on liver sections. Additionally, ABT-737 can reduce the myeloid colony growth and the LSK expansion in these mice; whilst abrogating BCL-2:RAS-GTP complex formation illustrated via biochemical and confocal assays. CONCLUSION: This represents the first in vivo progression model of MDS/AML dependent on the formation of a BCL-2:RAS-GTP complex rescued by ABT-737 via intrinsic apoptosis and thus support the case for BH-3 mimetic therapy.

Description: In almost all cases of acute promyelocytic leukemia (APL), t(15;17)(q22;q12) fuses the promyelocytic leukemia (PML) gene with the retinoic acid receptor alpha (RARA) gene. In rare cases of APL, other genes are fused to RARA including PLZF, NPM, NuMA, and STAT5b. These chimeras are collectively referred to as X-RARA fusions. Common features of the X-RARα fusions include their ability to form dimers through the X domain proteins while retaining the ability to interact with rexinoid receptors (RXRs, the proteins with which RARα normally heterodimerizes). We previously created an artificial X-RARα: p50-RARα (a fusion of the dimerization interface of p50 NFκB with the portion of RARα found in naturally occurring X-RARα proteins). p50-RARα has effects similar to PML-RARα including (i) enhancing repression at retinoic acid response elements, due to impaired release of co-repressors at low levels of all-trans retinoic acid (ATRA), and (ii) inhibiting differentiation of myeloid cell lines. We generated transgenic mice expressing p50-RARα under the control of the MRP8 promoter. p50-RARα had a minimal effect on myelopoiesis and initiated myeloid leukemias at a rate of

Description: Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D “knock-in” allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers.

Description: We showed previously that mild real hypoxia and hypoxia-mimetic agents induced in vitro cell differentiation of acute myeloid leukemia (AML). We here investigate the in vivo effects of intermittent hypoxia on syngenic grafts of leukemic blasts in a PML-RARα transgenic mouse model of AML. For intermittent hypoxia, leukemic mice were housed in a hypoxia chamber equivalent to an altitude of 6000 m for 18 hours every consecutive day. The results show that intermittent hypoxia significantly prolongs the survival of the leukemic mice that received transplants, although it fails to cure the disease. By histologic and cytologic analyses, intermittent hypoxia is shown to inhibit the infiltration of leukemic blasts in peripheral blood, bone marrow, spleen, and liver without apoptosis induction. More intriguingly, intermittent hypoxia also induces leukemic cells to undergo differentiation with progressive increase of hypoxia-inducible factor-1α protein, as evidenced by morphologic criteria of maturating myeloid cells and increased expression of mouse myeloid cell differentiation–related antigens Gr-1 and Mac-1. Taken together, this study represents the first attempt to characterize the in vivo effects of hypoxia on an AML mouse model. Additional investigations may uncover ways to mimic the differentiative effects of hypoxia in a manner that will benefit human patients with AML.