ALBERT

Description: FLT3 is an receptor tyrosine kinase of the subclass III family that plays a vital role in the regulation of the differentiation, proliferation and survival of normal hematopoietic cells. FLT3 mutations are often found in patients with Acute myelogenous leukemia (AML) and confer poor prognosis. Of these mutations, 15–35% are FLT3 ITD (internal tandem duplication) mutations and 5–7% are point mutations on the FLT3 kinase activation loop (e.g. D835V). Our laboratory is studying the signaling pathways associated with a newly identified multi-targeted tyrosine kinase receptor small molecule inhibitor (RTKI), ABT-869. Recently published work in our laboratory showed that using ABT-869 to treat MV4-11, a human AML FLT-3 ITD mutant cell line, resulted in the inhibition of phosphorylation of FLT-3 with a downstream inhibitory effect on the activation of STAT5, ERK, and Pim-1. Cell viability assays determined that MV-411 cells responded to ABT-869 in a concentration dependent manner (IC50 = 10nM). Apoptosis studies also showed an induction of apoptosis in ABT-869 treated cells. In vivo studies involving xenograft injections of MV-411 cells into SCID mice and subsequent treatment with ABT-869 demonstrated regression of tumor formation. In this study, a Ba/F3 mouse pro-B lymphocytic cell line harboring the FLT-3 ITD or FLT-3 D835V mutation is used as an isolated Flt-3 mutant model system. In vitro, ABT-869 is effective in inhibiting the proliferation of Ba/F3 Flt-3 ITD mutant cells when compared to Ba/F3 Flt-3 D835V mutant and Ba/F3 Flt-3 WT cells. Trypan Blue Exclusion and Alamar Blue assays were used to demonstrate that there is 50% inhibition of growth and proliferation (IC50) of Ba/F3 FLT3 ITD mutant cells at a concentration of 1nM after 48 hours of treatment. Ba/F3 FLT3 D835V mutant cells show an IC50 between 1μM and 10μM after 48 hours of treatment. In contrast, Ba/F3 FLT3 WT cells demonstrate an IC50 of 10μM only after 72 hours of treatment. Annexin V and propidium iodide staining of cells revealed that an increase in apoptosis (41.2%) occurred in Ba/F3 Flt-3 ITD mutant cells treated with 10nM ABT-869 after 24 hours when compared to untreated (6.5%) or vehicle control (6.1%) cells. Staining of Ba/F3 Flt-3 WT treated cell lines revealed no difference in apoptosis when compared to untreated Ba/F3 Flt-3 WT cell only and DMSO controls. PARP cleavage was observed in Ba/F3 FLT-3 ITD mutant cells following treatment with ABT-869 whereas no cleavage was observed with Ba/F3 WT cells treated with ABT-869. In vivo, the activity of ABT-869 treatment of SCID mice injected with Baf3 Flt-3 ITD, Baf3 Flt-3 D835V, or Baf3 Flt-3 WT cells is also being evaluated. Using bioluminescence imaging, it was determined that Ba/F3 FLT-3 ITD mutant and Ba/F3 Flt-3 D835Vmutant cell lines result in metastases and subsequent death in SCID mice after 2 weeks for ITD and 5 weeks for D835V, whereas mice injected with Ba/F3 WT survive longer than 5 weeks. Preliminary data demonstrated that ABT-869 prolonged survival in mice injected with the Ba/F3 FLT3-ITD cells compared to controls. Our preclinical data demonstrate that ABT-869 is effective specifically with FLT-3 ITD mutant cell lines in an isolated system. These studies provide rationale for the treatment of AML patients and the prevention of relapse.

Description: In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3–internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC50 approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC50 = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G0/G1 phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)–FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC50 approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC50 = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

Description: Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) receptor plays an important role in the pathogenesis of acute myeloid leukemia (AML). A number of small molecule kinase inhibitors are currently proceeding in different phases of clinical trials. As with imatinib in CML, leukemic cells could develop resistance to these RTK inhibitors when used as monotherapy. Mutations in the ATP-binding pocket have been identified through PCR-based mutagenesis screening in Ba/F3-FLT3-ITD cells and selected for growth in the presence of PKC412, or in a resistant Ba/F3-FLT3-ITD cell line developed by cocultured with SU5416. Resistance to PKC412 resulting from the N676K point mutation in the FLT3 kinase domain has been described in a clinical trial patient. Selection of activating Ras mutations has been found in 2 out of 6 FLT3 inhibitor resistant cell lines, but no point mutation of FLT3 kinase domain was found in all 6 resistant cell lines. To further investigate other potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R (IC50: 52 vs 6 nM for the parental MV4-11 cell line), which is also cross resistant to other structurally unrelated inhibitors including SU5416, AG1296 and a FLT3 inhibitor III (MERCK). No point mutation in the kinase domain of FLT3 was found in MV4-11-R cells. Western blot and FACS analysis excluded overexpression of p-FLT3, FLT3 or 3 multidrug resistance related proteins (MDR, MRP1 and LRP) in this resistant line. Gene expression profiling revealed up-regulation of FLT3 ligand (FLT3LG) (2.4 fold) and Survivin (2 fold), while down-regulation of SOCS1, SOCS2, and SOCS3 was observed in MV4-11-R compared to MV4-11 parental cells. Overexpression of FLT3LG and Survivin was also demonstrated at the protein level. Survivin is a unique member of the inhibitor of apoptosis proteins (IAP) family and a known target of the STAT3 pathway. Down-regulation of suppressor of cytokine signaling (SOCS) proteins (negative regulators of STAT pathways) was observed even in the presence of overactivation of the STAT1, STAT3 and STAT5 pathways in the MV4-11-R line. We screened a panel of small molecule inhibitors including a STAT inhibitor (indirubin derivative IDR E804), 3 JAK inhbitors (Tyrene CR4, AG490, and JAK3 Inhibitor II), and a CDK/survivin inhibitor (NU6140). We found that MV4-11-R is most sensitive to IDR E804 (an inhibitor of CDKs and the SRC-STAT3 pathway). The IC50 value of ABT-869 in MV4-11-R was decreased from 52 to 6.2 nM in the presence of 2 nM of IDR E804. Further validation of the therapeutic effect of IDR E804 in combination with ABT-869 in the MV4-11-R mouse xenograft model is ongoing. Targeting Survivin by shRNA and a dominant-negative vector (survivin-T34A) induced MV4-11-R to undergo apoptosis. Taken together, these results demonstrate that overactivation of STAT pathways and overexpression of survivin are the main mechanism of resistance to ABT-869; suggesting that the STAT pathways and survivin could be potential targets for the treatment of patients who develop resistance to FLT3 inhibitors.

Description: The early phase studies have shown the high response rates in chronic lymphocytic leukemia (CLL) patients treated with the BH3 mimetic venetoclax (ABT-199). It indicated that inhibition of BCL-2 is a viable strategy for the treatment of lymphoid malignancies. Objective anti-tumor responses were also observed in patients with other common B-cell non-Hodgkin lymphomas (NHLs) such as follicular lymphoma (FL) or diffuse large B-cell lymphoma (DLBCL), however the overall response rates are not as high as those in CLL patients. Targeting only one anti-apoptotic protein may lead to or uncover resistance owing to activity of other anti-apoptotic BCL2-family members in these settings. MCL-1 is associated with both intrinsic and acquired resistance to venetoclax and thus inhibition of MCL-1 is being explored through either direct inhibition or indirect targeting. Expression of MCL-1 is maintained via p-TEFb-mediated transcription, of which CDK9 plays a critical role. Here we aimed to investigate the combined effects of CDK9 inhibitor and venetoclax in primary DLBCL and FL cells. Inhibition of CDK9 via a small molecule A-1467729.0 (AbbVie) caused rapid loss in phosphorylation (Serine 2) of RNA polymerase II and MCL-1 expression in all tested primary cells of DLBCL and FL patients, confirming the intended effect of CDK9 inhibition. Primary samples from 12 NHL cases (6 DLBCL including 3 GCB/3 non-GCB and 6 FL) were tested for their ex vivo response to A-1467729.0 or venetoclax alone or in combination. Apoptosis assays showed negligible effects (

Description: All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

Description: To further investigate potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R. Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3 ligand increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by short-hairpin RNA (shRNA) in MV4-11-R cells induces apoptosis and augments ABT-869–mediated cytotoxicity. Overexpression of survivin protects MV4-11 from apoptosis. Subtoxic dose of indirubin derivative (IDR) E804 resensitizes MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R xenograft model. Taken together, these results demonstrate that enhanced activation of STAT pathways and overexpression of survivin are important mechanisms of resistance to ABT-869, suggesting that the STAT pathways and survivin could be potential targets for reducing resistance developed in patients receiving FLT3 inhibitors.

Description: Abstract 2617 Introduction: ABT-348 is a potent, novel, adenosine triphospate competitive inhibitor of Aurora A, B and C kinases. ABT-348 is also a potent inhibitor of all members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinase (RTK) families. ABT-348 has demonstrated strong antitumor activity in a variety of preclinical cancer cell line models, in vitro and in vivo, including leukemias as monotherapy and in combination with the DNA methyltransferase inhibitor, azacitidine. Objectives: The primary objectives of this study are to determine the safety and pharmacokinetics (PK) of orally and IV administered ABT-348 as monotherapy or in combination with azacitidine in patients (pts) with advanced hematologic malignancies. Secondary objectives include determination of the maximum tolerated dose (MTD) and exploration of biomarkers associated with ABT-348 activity. Methods: This phase 1 dose-escalation study of ABT-348 utilizes a modified continual reassessment method in pts with advanced hematologic malignancies: acute myelogenous leukemia (AML), acute lymphoblastic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia and myelodysplasia (MDS). Adult pts with histological or cytological confirmed disease, ECOG status 0–2, adequate hematological, renal and hepatic function and without significant hypertension or proteinuria are eligible. This study has four arms consisting of ABT-348 administered on a 28-day (D) cycle. Pts receive ABT-348 monotherapy once daily (QD, Arm A) or twice daily (BID; Arm B) on Days (D) 1, 8, and 15 of a 28-day cycle under fasting conditions. In Arm C, ABT-348 is given on the same schedule as Arm A in combination with azacitidine (75 mg/m2) administered IV or SC on Days 1 – 7 of each 28-day cycle. In Arm D, pts receive ABT-348 monotherapy on the same schedule as Arm A, but via IV administration. Samples are collected for PK and biomarker analyses. Pts are treated until progressive disease (PD) or unacceptable toxicity. Adverse event (AE) severity is graded using NCI-CTCAE v4.0. Results: 39 pts (median age, 66 y [45–86]) have enrolled (Arm A, n=32; Arm B, n=4; Arm C, n=3). Of these pts, 27 had AML, 11 had MDS and 1 had CML. Pts received a median of 4 prior therapies (range, 1 – 8). Best response to last prior therapy was: complete or partial response in 3 pts, stable disease in 11 pts, PD in 24, and not determined in 1. Dose escalation is shown in the table. The first dose-limiting toxicity (DLT) of grade 3 pancreatitis was observed at 640 mg in Arm A. Due to this DLT, along with a second case of grade 3 pancreatitis, further enrollment in Arm A was held and Arms B and C were opened. A DLT of grade 4 acute kidney injury was seen at 440 mg in Arm C. ABT-348-related AEs in greater than 2 pts were proteinuria (23%), nausea (21%), diarrhea (18%), hypertension (13%), vomiting (10%) and fatigue (8%). Grade 3/4 ABT-348-related AEs were hypertension (13%), pancreatitis (5%), acute kidney injury (3%) and diarrhea (3%). One patient in Arm A achieved a CRi (640 mg). Five of 32 pts in Arm A were treated for ≥4 cycles, including 2 of 7 pts at ABT-348 doses ≥ 640 mg. Based on preliminary PK analyses, Cmax and AUC of ABT-348 were approximately dose proportional in the dose range studied (10–690 mg); co-administration of azacitidine had no apparent effect on ABT-348 PK. Evidence of dual aurora/VEGF receptor kinase inhibition has been demonstrated by induction of polyploidy and PlGF, respectively. 33 pts have discontinued and 6 remain on study. Conclusions: ABT-348 has demonstrated on-target biomarker and clinical activity in pts with advanced hematologic magnancies, and was well tolerated at doses below 640 mg in Arm A. PK appears to be dose-proportional. Dose escalation is ongoing in Arms B and C (with azacitidine); an IV formulation of ABT-348 will also be evaluated. Disclosures: Off Label Use: Clofarabine in AML. Tibes:Abbott: Research Funding. Chiu:Abbott: Employment, Owns stocks Other. Xiong:Abbott: Employment, own Abbott stock Other. Qin:Abbott: Employment. Ansell:Abbott: Employment, own Abbott stock Other. Albert:Abbott: Employment, own Abbott stock Other. Tse:Abbott: Employment, own Abbott stock Other. Oliver:Abbott: Employment, own Abbott stock Other. Sajwani:Abbott: Employment, own Abbott stocks and participated in study design, data collection, analysis, interpretation and reviewing approving study documents related to ABT-348 programs. Other. McKee:Abbott: Employment, own Abbott stocks Other. Ricker:Abbott: Employment, own Abbott stocks Other.