Warner, Steven L.

Background:The expression of the receptor tyrosine kinase (RTK) Axl on cancer cells has been associated with poor prognosis and implicated in disease progression. Inhibition of Axl‐RTK with TP‐0903, a high affinity Axl‐RTK inhibitor induces robust apoptosis of several tumors, including chronic lymphocytic leukemia (CLL). Axl‐RTK inhibition has also been found to induce nonspecific immunomodulatory effects in syngeneic tumor models.Aims:We aimed to investigate the impact and downstream effects of Axl‐RTK inhibition on naïve T cells and chimeric antigen receptor T cells (CART), using CD19 directed CART cells (CART19) as a model.Methods:We utilized either PMA/Ionomycin or the CD19+ mantle cell lymphoma cell line, JeKo as a stimulant for naïve T cells and CART19, respectively. Cells were analyzed for cytokine profile, T cell phenotype, and kinetics after the stimulation in the presence or the absence of the Axl‐RTK inhibitor TP‐0903. Regarding in vivo experiment, a relapse JeKo xenograft model was used. Mice were treated with vehicle alone, TP‐0903 alone, CART19 alone, or TP‐0903 + CART19. Three weeks after the treatment, mice were re‐challenged with JeKo as a CART19 relapse model.Results:Stimulation of naïve T cells in the presence of the Axl‐RTK inhibitor TP‐0903 resulted in a significant reduction of Th2 cytokines and downregulation of inhibitory receptors. The differential expansion of either effector T cells or Tregs in the presence of TP‐0903 resulted in a preferential reduction of Tregs (Fig 1A). Axl‐RTK inhibition of CART19 led to Th1 polarization upon stimulation with the JeKo, as evident by the selective reduction of Th2 cytokine. Additionally, this resulted in a reduction of myeloid attracting cytokines, such as CXCL1, IL‐8, IL‐6, and IL‐10, cytokines implicated in the development cytokine release syndrome and neurotoxicity after CART cell therapy and in adaptive resistance to immunotherapy (Fig 1B). TP‐0903 also significantly downregulated inhibitory receptors on activated CARTs (Fig 1C). In our relapsed in vivo model, mice treated with CART19 + TP‐0903 rejected the JeKo challenge while mice received CART19 alone redeveloped disease, suggesting that Axl‐RTK inhibition enhanced CART cell persistence. Peripheral blood analysis of mice after the rechallenge showed a higher number of circulating T cells and lower level of MCP‐1. Western blotting was performed to investigate the downstream effect of Axl‐RTK inhibition on CART19 and it revealed a relatively selective inhibition of LCK on stimulated CART19. Transcriptome analysis of activated CARTs treated with TP‐0903 revealed >100 genes that were differentially expressed compared to untreated cells. Among these, Th cell and Treg polarization genes were significantly altered in activated CARTs treated with TP‐0903.imageFinally, we validated our findings in phase I clinical trial of TP‐0903 (NCT02729298). T cells isolated from 3 cancer patients showed a significant reduction of Tregs and Th1 polarization. This will be further investigated in a planned phase I clinical trial of TP‐0903 in relapsed/refractory CLL (NCT03572634).Summary/Conclusion:We showed for the first time that Axl‐RTK inhibition polarizes T cells into a Th1 functional phenotype, downregulates inhibitory receptors, and reduces myeloid associated cytokine production by CART19. The combination therapy yielded synergistic cytotoxicity against JeKo in vitro and in a CART19 relapse model. These findings encourage further study of Axl‐RTK inhibition with TP‐0903 as an enhancer of T cell immunotherapies.

Abstract The positive-transcription elongation factor (P-TEFb) complex is critical in stimulating the transcription of non-abortive transcripts by RNA polymerase II. The CDK9/cyclin-T heterodimer comprises the P-TEFb complex and promotes transcription elongation through phosphorylation of serine 2 of the heptapeptide repeats of the c-terminal domain of RNA Polymerase II (RNAPII-CTD). Alvocidib, a potent CDK9 inhibitor, has been shown to suppress expression of MCL-1 and Myc mRNAs, and clinical pharmacodynamic data suggest that alvocidib's activity is primarily mediated through the suppression of MCL-1 mRNA and protein expression. In addition to MCL-1 suppression, we hypothesized that CDK9 inhibition affects other RNA Polymerase II target RNAs, such as microRNAs. Recently, it was reported that inhibition of bromodomain and extraterminal domain (BET) protein by JQ1 suppresses miR17-92 expression. miR17-92 negatively regulates expression of the pro-apoptotic BH3-only protein, BIM, leading to suppression of BIM expression, thereby decreasing the cells' ability to induce apoptosis. It was hypothesized that CDK9 kinase activity is key to miR17-92 expression and suppression of BIM expression and that targeting CDK9 would lead to a decrease in miR17-92, and increases in BIM. MV4-11, OCI-AML3, MOLM13, and THP1 AML cell lines were used to determine the effects of alvocidib treatment on microRNAs. RT-qPCR was utilized to determine microRNA expression levels of miR17-92 and mRNA levels of BIM and other markers. Protein changes were determined using standard gel electrophoresis and immunoblotting technique. CellTiter-Glo and Caspase-Glo were used for all cell viability and apoptosis assays interrogating alvocidib. With CDK9 inhibition mediated via alvocidib, a dose- and time-dependent decrease in miR17-92 expression in MV4-11, OCI-AML3, MOLM13, and THP1 cells was observed. A decrease was observed even 3 hours post-treatment, persisting for up to 24 hours. MicroRNA suppression following treatment ranged in magnitude, with a maximal effect of between 3.3 to 4.2-fold suppression, depending on the cell line and timepoint. This suppression coincided with an increase in BIM mRNA and protein expression in the MV4-11, OCI-AML3, and MOLM13 cells. However, BIM protein increase was not observed in THP1 cells. Maximal increase in BIM mRNA levels reached 5.4-fold in the MV4-11 cell line. Our data suggest that CDK9 inhibition suppresses RNA Polymerase II-mediated expression of miR17-92, which in turn leads to increased expression of BIM. Combined with MCL-1 reduction, increased BIM protein expression mediated by alvocidib leads to enhanced apoptosis. Taken together, the data provide additional understanding of CDK9 as a potential therapeutic target, and are consistent with the hypothesis that CDK9 activity is necessary for miR17-92 expression. Citation Format: Hillary Haws, Hubert F. Arokium, James L. Bogenberger, Adam Siddiqui-Jain, David J. Bearss, Raoul Tibes, Steven L. Warner, Clifford J. Whatcott. Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 883.

Abstract Our study in CLL delineated a unique mechanism of how bone marrow stromal cells (BMSCs), may influence CLL B-cell signaling and drug resistance via modifying β-catenin and Axl status in leukemic cells. Purified primary CLL B-cells (n= 31) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=23) or CLL patients (n=12) 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 real-time PCRs. We observed significant increases in expression of Axl at 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 co-cultured CLL B-cells. But, we did not see any significant change in β-catenin or Axl expression in BMSCs co-cultured with CLL B-cells. The CLL B-cells from co-culture had an upregulation in downstream P-ERK-1/2 but no change in P-AKT(Ser473). High nuclear active-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) suggests inactivation of GSK-3β and β-catenin accumulation in co-cultured CLL B-cells. Additionally, we determined the phosphorylation status of Axl in CLL B-cells in co-culture compared with cultured alone. There was no change either at Y702 or total tyrosine phosphorylation 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 Axl inhibitor TP-0903 on CLL B-cells in co-culture vs cultured alone. Interestingly, both Axl and β-catenin expression levels were further upregulated in CLL B-cells exposed to TP-0903 compared to CLL B-cells without TP-0903 in co-culture, indicating possible roles for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance. However, TP-0903 decreased P-Axl(Y702) as well as an Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells even in presence of BMSCs. This was associated with induction of cell death and overrode BMSC mediated protection. Treatments of fludarabine or chlorambucil also led to increase in expressions of both β-catenin and Axl 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. Here we show that stroma mediated increased expression in both β-catenin and Axl likely contribute to the survival and drug resistance of B-cells from patients with CLL. Understanding their role in drug metabolism is underway. Citation Format: Sutapa Sinha, Charla Secreto, Justin Boysen, Steven L. Warner, David J. Bearss, Asish Ghosh, Neil E. Kay. Enhanced expression of β-catenin and Axl receptor tyrosine kinase in chronic lymphocytic leukemia (CLL) B-cells with co-culture on marrow stromal cells: Implications for drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2144.

Abstract 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 CLL plasma contained elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated functional implication of the FGF/FGFR signaling axis in CLL B cell biology. We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor, but not FGFR2/FGFR4, in primary CLL B cells. Interestingly, constitutively phosphorylated FGFR and its downstream signaling adaptor protein fibroblast receptor substrate 2-α were also detected in CLL B cells. However, in vitro stimulation of FGFR with recombinant bFGF was unable to increase endogenous phosphorylation levels of FGFR in CLL B cells from most CLL patients tested, suggesting existence of an autocrine FGF/FGFR signaling loop in the leukemic B cells. We know that Axl a known predominant RTK in CLL B cells seems to act as a docking site for multiple cellular kinases/lipase. Therefore, in an effort to determine whether Axl is functionally associated with FGFR in CLL B cells, we first examined if these two RTKs exist in the same molecular complex. Indeed we found that Axl forms a complex with FGFR3 in CLL B cells and is therefore likely to regulate FGFR signaling. Our hypothesis in this regard was further supported when we inhibited Axl phosphorylation in CLL B cells using a high-affinity Axl inhibitor (TP-0903; Tolero). Axl inhibition resulted in significant reduction of FGFR phosphorylation in CLL B cells. Finally, inhibition of FGFR signaling using a high-affinity FGFR inhibitor induced significant levels of apoptosis in CLL B cells (n=20) with a mean LD50 dose of ∼2.5 μM. Of interest, LD50 dose for CLL B cell apoptosis was much lower for the Axl inhibitor (mean LD50=140nM; n=20) compared to the FGFR inhibitor, which we believe occurs partly because TP-0903 seems to exert its inhibitory effect on P-FGFR likely via P-Axl inhibition in CLL B cells. In conclusion, we have detected expression of constitutively active FGFR in primary CLL B cells. Interestingly, our preliminary findings suggest that Axl forms an active RTK complex with FGFR and thus likely regulates FGFR signaling in these leukemic B cells. This is evidenced by the finding that Axl inhibition significantly reduced FGFR phosphorylation accompanied by massive induction of CLL B cell apoptosis, suggesting that targeting dual RTK signaling pathways (Axl and FGFR) in CLL B cells warrants further investigation as a way to develop a more effective and efficient therapeutic intervention for CLL patients. Citation Format: Sutapa Sinha, Justin Boyson, Charla Secreto, Steven L. Warner, Neil E. Kay, Asish K. Ghosh. Fibroblast growth factor receptor is expressed as a constitutively active receptor tyrosine kinase in chronic lymphocytic leukemia B cells and exists in an active complex with Axl: Dual targeting in CLL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3448. doi:10.1158/1538-7445.AM2014-3448

Abstract Purpose: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat patients with CLL. Experimental Design: Expression/activation status of other members of the TAM (e.g., Tyro3, Axl, and MER) family of RTKs in CLL B cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without the presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on the Axl signaling pathway were also evaluated in CLL B cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination. Results: CLL B cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and forms a complex with Axl in CLL B cells. TP-0903 induces massive apoptosis in CLL B cells with LD50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B cells from TP-0903–induced apoptosis. A marked reduction of the antiapoptotic proteins Mcl-1, Bcl-2, and XIAP and upregulation of the proapoptotic protein BIM in CLL B cells was detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis. Conclusions: Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating patients with CLL. Clin Cancer Res; 21(9); 2115–26. ©2015 AACR.