ALBERT

Description: Introduction CLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance. Methods Purified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture. Results We observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture. Conclusions Here we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Description: Discovery of somatic mutation of JAK-2 (G1849T that produces JAK-2V617F) in the hematopoietic cells of patients with Philadelphia chromosome negative myeloproliferative disorders (Ph−MPDs) was a watershed event that not only provided new insights into the pathobiology of polycythemia vera, essential thrombocytosis and primary myelofibrosis but also identified a potential target for therapy. Herein we report the results of preclinical studies designed to characterize the activity of a novel inhibitor of JAK-2. The compound, SGI-1252, developed by SuperGen (Dublin, CA) incorporates with high affinity into the ATP-binding site of JAK-2. SGI-1252 was tested against a panel of 75 kinases and was found to have significant activity against only FLT-3, TYK-2 and the SRC family members, ABL, LCK, YES, in addition to JAK-2 and JAK-1. SGI-1252 has an IC50 for JAK-2 of 5.4 nM with an IC50 for JAK-2V617F of 19.7 nM. The inhibitor also effectively blocks the activity of JAK-1 (IC50 14.8 nM) but has little JAK-3 inhibitory activity (IC50 1,700 nM). SGI-1252 is a potent inhibitor of STAT-5 phosphorylation (EC50 76.2 nM) and was also found to block the JAK-2 dependent expression of the anti-apoptotic protein, BCL-XL (EC50 778 nM). Drug treatment of a murine cell line (FDCP) transfected with either human wild-type JAK-2 or JAK-2G1849Tgenerated IC50 values of 83 nM and 108 nM, respectively, and SGI-1252 treatment of human cell lines, HEL, UKE-1 and SET-2, that express mutant JAK2 in different copy numbers, gave IC50 values of 472 nM, 83 nM and 63 nM, repectively. When tested in ex-vivo expanded native human erythroid progenitor cells from 17 patients with Ph−MPDs (10 PV and 7 MF), SGI-1252 showed an IC50 of ~100 nM, regardless of the JAK-2V617F allele burden. Using a flow cytometric assay, SGI-1252 was shown to induce apoptotic cell death in a concentration dependent manner. Luminex technology allows for concurrent quantitative analysis of multiple proteins from the same tissue source, and this technology was used to investigate simultaneously the effects of SGI-1252 on total and phospho ERK1/2, total and phospho STAT3, phospho STAT5, caspase 3, cleaved PARP and GAPDH (control) in untreated and drug treated cells at IC50 and IC80 concentrations. Significant in vivo efficacy of SGI-1252 was also observed using HEL and MV-4-11 xenograft models when compared to treatment with vehicle or daunorubicin. Using a murine model, we found that SGI-1252 has high oral bioavailability and is well tolerated with a five-day repeat maximum dose of at least 900 mg/kg. Together, these studies demonstrate that SGI-1252 is a potent inhibitor of JAK-2 dependent proliferation in both JAK-2V617F positive cell lines and in ex vivo expanded erythroid progenitors derived from patients with JAK-2V617F positive Ph−MPDs. Moreover, our studies show that the effects of SGI-1252 are mediated by blocking both JAK-2 dependent anti-apoptoic pathways and JAK-2 dependent proliferative pathways. Using the orally available form of the compound, pharmacokinetic, pharmacodynamic and toxicity studies in mice suggest that serum concentration of the drug well above the predicted therapeutic range can be achieved without significant hematological toxicity. Based on these preclinical experiments, SGI-1252 appears to be an excellent candidate for phase I/II studies in patients with Ph−MPDs.

Description: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells. Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells. To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition. In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Description: Despite significant efforts, the clinical mechanism of action of hypomethylating agents such as 5-azacytidine (5-aza) is still poorly understood. 5-aza is currently indicated for the treatment of patients with myelodysplastic syndrome (MDS). While 5-aza has achieved good single-agent activity in acute myeloid leukemia (AML), complete response rates remain low when used as a single agent. In a recent report aimed at identifying rational therapeutic combinations with 5-aza, Bogenberger and colleagues identified multiple BCL-2 family member/BH3-containing therapeutic targets, which synergize with 5-aza when inhibited genetically or pharmacologically. The CDK9 inhibitor, alvocidib, has achieved significant improvement in complete response rates of newly diagnosed AML patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized multi-center Phase 2 trial when compared to 7+3 standard of care treatment. Recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the pro-apoptotic activity of alvocidib in the FLAM regimen. We, therefore, hypothesized that alvocidib and 5-aza would synergize therapeutically in the treatment of AML by means of transcriptional repression of MCL-1 and sensitization to 5-aza. In this report, we demonstrate that treatment of AML cell lines with alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion. Pre-treatment of cells with alvocidib, to repress MCL-1 expression prior to 5-aza treatment, reduced the 5-aza cell viability EC50 more than 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/5-aza combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. Therefore, following reports suggesting inhibition of BCL-2 family members including MCL-1, sensitizes cells to 5-aza, our data suggest that the alvocidib/5-aza combination may constitute a viable therapeutic regimen. We also conclude that a CDK9 inhibitor/5-aza combination may be an effective clinical approach for the treatment of AML. Disclosures Kim: Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Description: Abstract 2758 Poster Board II-734 Axl kinase, a member of the TAM family and also known as UFO, ARK, and Tyro7, is a receptor tyrosine kinase implicated in tumorigenesis. Overexpression of Axl is associated with increased cellular transformation, cell survival, proliferation, migration, angiogenesis, and adhesion. The oncogenic potential of Axl was first discovered in chronic myelogenous leukemia (CML) and has been shown to play a role in the development of acute myelogenous leukemia (AML) and myelodysplasia. Binding of Growth Arrest Specific Gene-6 (GAS6), a vitamin K-dependent protein and a known ligand for Axl, leads to subsequent phosphorylation events of downstream effecter molecules such as mitogen-activated protein (MAP) kinase and phosphatidyl-inositol (PI)-3 kinase/AKT pathways which are critical in oncogenic transformation. Furthermore, target validation studies of in vivo cancer models show that inhibition of Axl expression by RNA interference blocked tumor growth in those models. Taken together, this information makes Axl kinase an exciting target for small molecule drug discovery. Effectively utilizing SuperGen's proprietary CLIMB“technology, a series of small molecule inhibitors were rapidly discovered and developed for potency and selectivity against Axl. Lead candidate compounds demonstrate low nano-molar IC50 values in an Axl kinase biochemical assay, and focused selectivity for Axl when screened in kinase panels. Likewise, these compounds showed low and sub micro-molar activity from an anti-proliferative leukemia/lymphoma cell-based panel. Similarly, treatment of leukemia cells with these compounds in combination with known chemotherapeutic agents produced results that implicate combinatorial therapies as potentially beneficial treatments for patients suffering from hematological malignancies. In mechanistic, target validation assays, EC50 data from immunoprecipitated western blots of transiently transfected liquid tumor cells showed nano-molar inhibition of phospho-Axl when detecting with an anti-phospho-tyrosine antibody. A western blot panel of leukemia and lymphoma cell lines showed overexpression of Axl in several of these cell lines, further validating Axl as a potential therapeutic target in hematological cancers. Functional cell-based assays using SuperGen's lead compounds also potently inhibited phospho-AKT (S473). We propose that SuperGen's small molecule inhibitors against Axl kinase represent a new class of compounds with potent and selective activity in hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Alvocidib has demonstrated a significant improvement in the complete response rates of newly diagnosed acute myeloid leukemia (AML) patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized Phase 2 study compared to 7+3, the current standard of care. Although the mechanism of action of alvocidib as a single agent is documented, the mechanism underlying synergy found in the FLAM regimen is still not fully understood. The FLAM regimen was originally developed based on the perceived benefit of time-sequential cell cycle arrest (alvocidib) followed by release of the cells from cell cycle arrest and inhibition of DNA replication (cytarabine) during S-phase. However, recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the activity in the FLAM regimen. We, therefore, hypothesized that MCL-1 transcriptional repression constitutes the primary mechanism for the synergism observed with the treatment of the FLAM regimen. Here, we demonstrate that treatment with alvocidib, followed by treatment with cytarabine and mitoxantrone, is synergistic in vitro and correlates with the downregulation of MCL-1 expression. The FLAM regimen results in significant increases in caspase activity in comparison to any single agent within the combination. As has been previously reported, we also observe that increased activity of cytarabine in alvocidib-treated cells corresponds with progression into the S-phase of the cell cycle, following the washout of alvocidib. However, this observation accounts for only a small portion of the inhibition of cell proliferation. This is further confirmed by the observation that CDK4/6 (cell cycle) specific inhibitors, such as palbociclib, do not show synergistic increases in caspase activity following treatment in the same setting. In various AML cell lines treated with MCL-1 siRNA, followed by cytarabine and mitoxantrone treatment, we also observe a synergistic increase in the inhibition of cell proliferation. Therefore, considering our earlier work showing that MCL-1 dependence predicts AML patient response to the FLAM regimen, we propose that MCL-1 repression is the primary mechanism of alvocidib's biological activity and also a primary mechanism conferring resistance to cytarabine. We also conclude that the FLAM regimen is an effective regimen, clinically, in treating patients with high-risk AML, as a consequence of its inhibition of transcription via CDK9. Disclosures Kim: Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Lee:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Description: Mesenchymal stem cells (MSCs) contribute to the regeneration of mesenchymal tissues, and are essential in providing support for the growth and differentiation of primitive hemopoietic cells within the bone marrow microenvironment. It is becoming increasingly clear that the tumor microenvironment plays a very important role in tumor progression and drug resistance, and the selection of cancer cells possessing the mesenchymal phenotype leads to drug resistance in many different tumor types. We have been exploring the role of the protein Axl in promoting the mesenchymal phenotype in both myeloid and lymphoid malignancies, and the role of Axl in promoting drug resistance in these malignancies. The signaling downstream of Axl that leads to the acquisition of the mesenchymal phenotype has not been well elucidated. Following results from a genetic screen using a zebrafish model, we have discovered a role for retinoic acid (RA) signaling which is regulated by Axl and controls the mesenchymal phenotype in leukemic cells. In addition, recent reports have shown an interaction between a retinoic acid regulated gene, RARRES1, and Axl, leading our group to seek to understand the role of retinoic acid signaling in the control of AXL. We hypothesized that treatment with our AXL inhibitor, TP-0903, would disrupt RA signaling and lead to a reversal of the mesenchymal phenotype in leukemia cells. Following TP-0903 treatment, we interrogated changes in mRNA expression using RT-PCR, protein expression using standard immunoblotting, and endogenous RA levels using a competitive ELISA. We also assessed the effect of TP-0903 on tumor growth in an in vivo model, assessing efficacy of TP-0903 in an MV4-11 xenograft mouse model. One of the genes that we detected being dramatically changed by treatment with TP-0903 was the RA metabolizing protein CYP26A1, suggesting that Axl inhibition indeed leads to changes in RA metabolism. We observed a strong induction of CYP26 mRNA expression following RA treatment in MV4-11 leukemia cells which was also observed in treatment with our AXL inhibitor, TP-0903, at levels as low as 100 nM. We also assessed TP-0903 activity in additional cell lines (HL60, A549, and H1650), and with an alternative AXL inhibitor, R428. Importantly, TP-0903 treatment correlated with increased CYP26 expression and reduced levels of endogenous RA. In vivo, TP-0903 strongly inhibited xenograft tumor volumes by up to 100% with multiple dose levels and treatment schedules. CYP26 expression in fixed tissues correlated well with mRNA levels observed in xenograft tumors following treatment. Taken together, our observations support our hypothesis that inhibition of AXL kinase by TP-0903 can disrupt RA metabolism by inducing CYP26 expression and this disruption of RA metabolism leads to reversal of the mesenchymal phenotype in leukemic cells. Disclosures Soh: Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Description: Introduction: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite that still requires options for treatment despite recent advances with BTK therapy. We previously found that Axl receptor tyrosine kinase (RTK), a member of the TAM (Tyro3, Axl, MER) family of RTKs, plays a critical role in CLL B-cell survival (Blood 2011, 117:1928). Here, we explored the levels of apoptosis induction of CLL B-cells using a novel high-affinity Axl inhibitor as a single agent or in combination with BTK inhibitors. Methods: Expression/activation status of the Tyro3, Axl and MER in CLL B-cells was evaluated by Immunoprecipitation/Western blots. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 (Tolero Pharmaceuticals) or FGFR inhibitor TKI-258 (Novartis) with or without the presence of CLL bone marrow stromal cells (BMSCs). Impact of Axl or FGFR signal inhibition on CLL B-cell survival was also evaluated by flow cytometry after staining with annexin V/propidium iodide. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination in induction of apoptosis. Combination index (CI) of two agents was calculated using the method of Chou and Talalay, 1984. Results: Although we defined pro-survival role of Axl in CLL B-cells, contribution of other members of the TAM family of RTKs including Tyro3 and MER to prolonged survival of the leukemic B-cells remains largely undefined. In this study we found that CLL B-cells overexpress Tyro3, but not MER, and that Tyro3 is constitutively phosphorylated. Interestingly, immunoprecipitation of Tyro3 from CLL B-cell lysates shows retention of highly active Axl in the immune complex suggesting that these two RTKs either heterodimerize or form a molecular complex in CLL B-cells. Our earlier work found that Axl regulates multiple cell survival signaling pathways including PI3K/AKT and Src and that, Axl inhibition using a first generation Axl inhibitor induced apoptosis in CLL B-cells (Blood 2011, 117:1928). Thus, we hypothesized that targeting Axl using a very high affinity Axl inhibitor would induce even higher levels of apoptosis in CLL B-cells compared to the first generation Axl inhibitor. To address this, we utilized a high-affinity orally bioavailable Axl inhibitor TP-0903 to generate preliminary information on its efficiency to specifically target Axl and the level of apoptosis induction in CLL B-cells. Indeed TP-0903 as a single agent induced a robust apoptotic cell death in CLL B-cells within 24 hours of exposure at nanomolar doses, which are easily achievable in vivo (information provided by Tolero Pharmaceuticals). Interestingly, while TP-0903 effectively reduces phosphorylation on Axl in CLL B-cells, it was unable to target P-Tyro3 suggesting that Axl is likely the predominant RTK of the TAM family in CLL B-cells. A marked reduction of the anti-apoptotic proteins Mcl-1, Bcl-2, XIAP and upregulation of the pro-apoptotic protein BIM in CLL B-cells via activation of its upstream regulator FOXO3a were noted as a result of inhibition of P-Axl and its downstream signaling pathways, P-AKT/P-Src. Importantly, TP-0903 was able to overcome CLL BMSC mediated protection of the leukemic B-cells from apoptosis induction. Given the observed robust induction of apoptosis with TP-0903 and the known impact of BTK inhibitors on CLL patient clinical outcome, we then exposed CLL B-cells to TP-0903 in combination with two BTK inhibitors that included Ibrutinib and a reversible BTK inhibitor TP-4216 (a kind gift from Tolero Pharmaceuticals). Based on the CI values, we found that the in vitro combination of TP-0903 and Ibrutinib showed additive effects in CLL B-cells from 6 of 11 CLL patients, CLL B-cells from the remaining 5 CLL patients showed antagonistic effects (Fig. 1A). However, we observed synergistic effects of TP-0903 in combination with TP-4216 in the induction of apoptosis in CLL B-cells from 2 of 10 patients and additive effects in CLL B-cells from 6 patients (Fig. 1B). Cells from the remaining 2 patients exhibited a slightly antagonisticeffect. Conclusion: In total, these results suggest that TP-0903 is quite effective in inducing robust CLL B-cell apoptosis as a single agent even at a nanomolar concentration and its combination with the reversible BTK inhibitor TP-4216 could be an effective and unique strategy for the treatment of CLL patients. Figure 1 Figure 1. Disclosures Warner: Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Bearss:Tolero Pharmaceuticals: Employment. Shanafelt:Genentech: Research Funding; GlaxoSmithKline: Research Funding; Celegene: Research Funding; Cephalon: Research Funding; Pharmacyclics/Jannsen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Description: Anemia of chronic disease (ACD) is an inflammatory cytokine driven disease characterized by hypoferremia despite adequate iron stores. This is largely due to hepcidin, a master regulator of iron homeostasis, which blocks enterocytes from absorbing iron and preventing iron release from macrophages by binding to ferroportin. It is known that bone morphogenetic proteins (BMP) up-regulate hepcidin by activating the SMAD signaling pathway through the activin-like kinase receptor 2 (ALK2). Therefore, ALK2 has emerged as a potential therapeutic target to modulate hepcidin levels and treat ACD. We have developed a novel series of small molecule ALK2 inhibitors with promising activity in preclinical models of ACD. Using well-established cell-based and animal models of hepcidin signaling and anemia, we optimized and validated the activity of the most promising preclinical lead candidates. These compounds demonstrate significant activity in downregulating hepcidin expression in BMP-induced cell culture studies at concentrations of 100 nM or lower. Importantly, this hepcidin lowering activity was observed at concentrations that exhibited no cytotoxicity suggesting the compounds have a clean selectivity profile. The compounds also demonstrated remarkable activity in animal models of anemia, including an acute model induced by the administration of turpentine oil and a more chronic model induced by tumor formation and growth. Treatment with the lead candidates completely reversed the induction of hepcidin expression in these models and also decreased the symptoms of anemia as measured by serum iron and red blood cell levels. From these data, we have nominated a candidate to advance into IND-enabling studies that has favorable drug-like properties. We anticipate a clinical development strategy that focuses on anemia of cancer with subsequent expansion into anemia associated more broadly with other inflammatory and chronic diseases Disclosures Kim: Tolero Pharmaceuticals: Employment. Maughan:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Description: In individuals with chronic inflammatory diseases, such as cancer or rheumatoid arthritis, constitutive signaling through ALK2, a member of the bone morphogenetic protein (TGFβ/BMP) receptor family, leads to debilitating anemia, commonly referred to as anemia of chronic disease (ACD). Activation of ALK2, like other members of the BMP receptor family, leads to the phosphorylation and activation of SMAD family transcription factors via signal transduction and subsequent activation of gene expression. Activation of ALK2 in the liver induces the SMAD-driven transcription of the peptide hormone hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Lowering constitutively elevated hepcidin levels by inhibiting ALK2 kinase activity is a potentially viable therapeutic strategy for ACD. Current therapeutic approaches for ACD rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which address the underlying pathological deficit of functionally low iron levels. TP-0184 is a small-molecule, selective inhibitor of ALK2 kinase activity (IC50 = 5 nM). TP-0184 has demonstrated profound preclinical activity in three mouse efficacy models for ACD. In model 1, TP-0184 reversed hepcidin induction in mice treated with turpentine oil. In model 2, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection with heat-inactivated Brucella abortus. In model 3, TP-0184 reversed elevated hepcidin levels in TC-1 tumor bearing mice. Plasma and liver pharmacokinetics in mice revealed that TP-0184 has a high volume of distribution (Vd = 30.8) and accumulates at high concentrations in the liver (Cmax of 292 mM following a single oral dose of at 20 mg/kg). In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Collectively these studies support the clinical evaluation of TP-0184 as an alternative treatment for ACD. Disclosures Peterson: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.