Frank Frissora

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

Tumor antigen ROR1 targeted drug delivery mediated selective leukemic but not normal B-cell cytotoxicity in chronic lymphocytic leukemia.

Selective cytotoxicity to cancer cells without compromising their normal counterparts pose a huge challenge for traditional drug design. Here we developed a tumor antigen-targeted delivery of immunonanoparticle carrying a novel non-immunosuppressive FTY720 derivative OSU-2S with potent cytotoxicity against leukemic B cells. OSU-2S induces activation of protein phosphatase 2A (PP2A), phosphorylation and nuclear translocation of SHP1S591 and deregulation of multiple cellular processes in chronic lymphocytic leukemia (CLL) resulting in potent cytotoxicity. To preclude OSU-2S-mediated effects on these ubiquitous phosphatases in unintended cells and avoid potential adverse effects, we developed an OSU-2S-targeted delivery of immunonanoparticles (2A2-OSU-2S-ILP), that mediated selective cytotoxicity of CLL but not normal B cells through targeting receptor tyrosine kinase ROR1 expressed in leukemic but not normal B cells. Developing a novel spontaneous CLL mouse model expressing human ROR1 (hROR1) in all leukemic B cells, we demonstrate the therapeutic benefit of enhanced survival with 2A2-OSU-2S-ILP in vivo. The newly developed non-immunosuppressive OSU-2S, its delivery using human CLL directed immunonanoparticles and the novel transgenic (Tg) mouse model of CLL that expresses hROR1 exclusively in leukemic B cell surface are highly innovative and can be applied to CLL and other ROR1+ malignancies including mantle cell lymphoma and acute lymphoblastic leukemia.

IFN-γ-mediated inhibition of antigen receptor-induced B cell proliferation and CREB-1 binding activity requires STAT-1 transcription factor

We report here a role for cyclic AMP‐responsive element‐binding protein‐1 (CREB‐1) in B cell antigen receptor (BCR)‐induced growth inhibition by IFN‐γ. BCR‐induced proliferation is negatively regulated by IFN‐γ. Stimulation through BCR resulted in dose‐dependent induction of CREB‐1 binding to the consensus cyclic AMP‐responsive element. Recombinant IFN‐γ inhibited the BCR‐induced CREB‐1 DNA binding activity and cell proliferation in B cells from signal transducer and activator of transcription‐1 (STAT‐1)+/+, but not STAT‐1–/– mice. These studies provide the first evidence for cross‐talk between the STAT‐1 and CREB‐1 signaling pathways in IFN‐γ‐mediated negative regulation of B cell activation.

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model.

The gene encoding PTPROt (truncated isoform of protein tyrosine phosphatase receptor-type O) is methylated and suppressed in chronic lymphocytc leukemia (CLL). PTPROt exhibits in vitro tumor-suppressor characteristics through the regulation of B-cell receptor (BCR) signaling. Here we generated transgenic (Tg) mice with B-cell-specific expression of PTPROt. Although lymphocyte development is normal in these mice, crossing them with TCL1 Tg mouse model of CLL results in a survival advantage compared with the TCL1 Tg mice. Gene expression profiling of splenic B-lymphocytes before detectable signs of CLL followed by Ingenuity Pathway Analysis revealed that the most prominently regulated functions in TCL1 Tg vs non-transgenic (NTg) and TCL1 Tg vs PTPROt/TCL1 double Tg are the same and also biologically relevant to this study. Further, enhanced expression of the chemokine Ccl3, the oncogenic transcription factor Foxm1 and its targets in TCL1 Tg mice were significantly suppressed in the double Tg mice, suggesting a protective function of PTPROt against leukemogenesis. This study also showed that PTPROt-mediated regulation of Foxm1 involves activation of p53, a transcriptional repressor of Foxm1, which is facilitated through suppression of BCR signaling. These results establish the in vivo tumor-suppressive function of PTPROt and identify p53/Foxm1 axis as a key downstream effect of PTPROt-mediated suppression of BCR signaling.

ROR1-targeted delivery of OSU-2S, a nonimmunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle-cell lymphoma in vitro and in vivo

Mantle-cell lymphoma (MCL) remains incurable despite numerous therapeutic advances. OSU-2S, a novel nonimmunosuppressive FTY720 (Fingolimod) derivative, exhibits potent cytotoxicity in MCL cell lines and primary cells. OSU-2S increased the surface expression of CD74, a therapeutic antibody target in MCL cells. OSU-2S, in combination with anti-CD74 antibody milatuzumab, enhanced cytotoxicity in MCL. Moreover, MCL tumor antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeted immunonanoparticle-carrying OSU-2S (2A2-OSU-2S-ILP)-mediated selective cytotoxicity of MCL in vitro, as well as activity in a xenografted mouse model of MCL in vivo. The newly developed OSU-2S delivery using ROR1-directed immunonanoparticles provide selective targeting of OSU-2S to MCL and other ROR1(+) malignancies, sparing normal B cells.

Modulation of Ets-1 expression in B lymphocytes is dependent on the antigen receptor-mediated activation signals and cell cycle status.

In this report, we tested the hypothesis that Ets‐1 transcription factor is modulated at the mRNA level during B cell antigen receptor (BCR)‐induced cell‐signalling events. Quiescent B cells express high levels of Ets‐1 mRNA. Stimulation through the BCR results in time‐dependent inhibition of Ets‐1 mRNA expression in primary splenic B cells with maximal inhibition observed by 16‐h post‐stimulation. Inhibition of Ets‐1 expression is specific to antigen receptor but not CD40‐mediated activation. Antigen receptor–induced inhibition of Ets‐1 mRNA can be mimicked by phorbol myristate acetate (PMA) and/or ionomycin. PMA but not ionomycin‐induced inhibition of Ets‐1 expression is rescued by the inhibitors of protein kinase C and MEK. Extended time‐course analysis revealed a time‐dependent cyclical pattern in the re‐expression of Ets‐1 mRNA. While resting cells revealed maximal Ets‐1 mRNA expression, activation events that induced exit from G0/G1 or cells blocked in early S phase exhibited decreased Ets‐1 mRNA levels. Interestingly, cells arrested at late G2 or M phase of the cell cycle failed to down modulate Ets‐1 mRNA expression. Overexpression of Ets‐1 in 70Z/3 B cell line caused abnormal accumulation of cells in S phase associated with increased cyclin A expression. Consistent with a requirement for Ets‐1 in BCR‐induced cell cycle entry, splenic B cells from mice deficient in Ets‐1 showed defective antigen receptor–induced DNA synthesis and S phase entry. These results suggest a critical role for Ets‐1 regulation during B cell activation and cell cycle entry.

OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway

Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU‐DY7, a novel D‐tyrosinol derivative targeting p38 mitogen‐activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC‐1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose‐ and time‐dependent manner. The OSU‐DY7‐induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase‐3 activation and poly (ADP‐ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z‐VAD‐FMK. Interestingly, OSU‐DY7‐induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B‐CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU‐DY7‐mediated cytotoxicity. Furthermore, OSU‐DY7‐induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU‐DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.

Abstract 4406: ROR1 targeted delivery of OSU-2S, a non-immunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle cell lymphoma in-vitro and in-vivo

Treatment of mantle cell lymphoma (MCL), an uncommon non-Hodgkin9s lymphoma, remains challenging despite numerous therapeutic advances. We have previously shown the preclinical effect of FTY720 (Fingolimod) against MCL through down modulation of cyclin D1, the protein almost uniformly over-expressed in this disease. Herein, we describe the potent direct cytotoxicity of OSU-2S, a novel non-immunosuppressive FTY720 derivative in MCL cells and evaluate tumor directed lipid based nanoparticle formulation of OSU-2S designed to selectively deliver to ROR1+ MCL cells. OSU-2S is a FTY720 derivative that does not traffic T cells and exhibits potent cytotoxicity in MCL cell lines and in MCL patient-derived primary cells (p = 0.0049). Exploratory studies aimed to identify the best combination therapies identified induction of cell surface CD74 in primary MCL cells treated with OSU-2S. Similarly, induction of cell surface CD74 was also confirmed in JeKo and Mino cells. Evaluation of OSU-2S and anti-CD74 antibody, milatuzumab in cell lines and primary MCL cells revealed enhanced cytotoxicity compared to either of the agents alone. B-cell malignancy restricted expression of receptor tyrosine kinase ROR1 in MCL, chronic lymphocytic leukemia (CLL) and subset of pediatric acute lymphocytic leukemia (ALL) has been reported. Consistent with this, JeKo, Mino and primary MCL cells but not normal B cells expressed ROR1. As tumor directed delivery of cytotoxic cargo offers the potential to further enhance the therapeutic index of cancer therapeutics such as OSU-2S, we developed a lipid-based OSU-2S nanoparticle (OSU-2S-LP) tethered with an anti-ROR1 mouse monoclonal antibody (2A2) to form 2A2-OSU-2S-ILP that mediated selective cytotoxicity of MCL. Testing of immunonanoparticle formulation on JeKo, Mino cell lines and primary lymphoma cells showed selective cytotoxicity of 2A2-OSU-2S-ILP in MCL (p −/− mice revealed significantly diminished tumor growth compared to the control (p = 0.0001). These findings describe the novel OSU-2S molecule as a successor of FTY720 that is active against MCL. OSU-2S enhanced CD74 expression can be exploited in combination therapy with milatuzumab which is in human clinical trials. Importantly, the tumor antigen ROR1 directed delivery system for increasing the payload selectively to the cancer cells thus obviating the exposure of chemotherapeutics and targeted therapeutics to other unintended target cells has broad implications in diverse ROR1+ malignancies. Citation Format: Rajeswaran Mani, Chi-Ling Chiang, Frank W. Frissora, Ribai Yan, Xiaokui Mo, Sivasubramanian Baskar, Christoph Rader, Mitch Phelps, Ching-Shih Chen, Robert Lee, John Byrd, Robert Baiocchi, L James Lee, Natarajan Muthusamy. ROR1 targeted delivery of OSU-2S, a non-immunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle cell lymphoma in-vitro and in-vivo . [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4406. doi:10.1158/1538-7445.AM2015-4406

Abstract 492: Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression.

Interleukin-2 Inducible Kinase (ITK) is an appealing therapeutic target given its critical role in polarizing T-cell immunity against various infectious, autoimmune, and neoplastic diseases. ITK activity promotes Th2 immunity, thereby subverting healthy Th1-based immune surveillance. Th2 CD4 T-cells depend on ITK for activation, whereas Th1 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) that conducts T-cell receptor activation in the absence of ITK. Similar to many other tumors, chronic lymphocytic leukemia (CLL) directs an aberrant Th2 bias that drives immune evasion, promotes a supportive microenvironment, and cripples adaptive immunity. In CLL, this immune suppression leads to severe infections which are a leading cause of death. Although ITK inhibitors have the promise of alleviating tumor-induced immune suppression, to date no clinically viable ITK inhibitor exists. Ibrutinib is an irreversible inhibitor of Bruton9s tyrosine kinase (BTK) with outstanding activity and tolerability in B-cell malignancies. The striking homology between BTK and ITK combined with our in silico docking studies and in vitro kinase inhibition profiles led us to identify ibrutinib as the first clinically viable irreversible ITK inhibitor. We further confirmed both molecular and functional outcomes using Jurkat T-cells and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation, offering a molecular explanation for the selective outgrowth of cytotoxic Th1-biased immunity. This effect was confirmed using T-cells directly derived from CLL patients. To clearly demonstrate this effect in vivo, we utilized the Leishmania major model of Th2-mediated infection. Ibrutinib enhanced resistance to the parasite by eliciting robust Th1 immunity. To demonstrate a direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model, we observed complete recovery of functional immunity, with all ibrutinib-treated mice surviving a potentially lethal Listeria monocytogenes infection. We further confirmed irreversible binding of ITK in patients currently receiving ibrutinib as part of a clinical trial. Finally, we found that ibrutinib inhibited phosphorlyation of ITK9s direct downstream target PLCγ1 in CD4 T-cells. Together, our results provide novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib9s irreversible ITK inhibitory effects may prove beneficial in a number of other autoimmune, inflammatory, parasitic, and viral diseases. Citation Format: Jason A. Dubovsky, Kyle A. Beckwith, Gayathri Natarajan, Jennifer A. Woyach, Samantha Jaglowski, Joshua Hessler, Ta-Ming Liu, Betty Y. Chang, Karilyn M. Larkin, Matthew R. Stefanovski, Frank W. Frissora, Lisa L. Smith, Kelly A. Smucker, Joseph M. Flynn, Jeffrey A. Jones, Leslie A. Andritsos, Kami Maddocks, Amy M. Lehman, Richard Furman, Jeff Sharman, Anjali Mishra, Michael A. Caligiuri, Abhay R. Satoskar, Joseph J. Buggy, Natarajan Muthusamy, Amy J. Johnson, John C. Byrd. Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2013-492

Abstract 1351: Analysis of the in vivo tumor suppressive potential of PTPROt (truncated protein tyrosine phosphatase receptor-type O) in CLL using a transgenic mouse model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Protein tyrosine phosphatases (PTPs) and kinases (PTKs) and their corresponding substrates are integrated within elaborate signal transduction networks essential for regulating several important cellular functions including growth, differentiation, cell cycle, gene transcription, the immune response and survival. Defective operation of these networks leads to aberrant tyrosine phosphorylation that contributes to the development of many human diseases including cancer. Importantly, as observed with PTKs, deregulation of PTPs plays a key role in the pathogenesis of these diseases. Thus, PTPs and their substrates represent novel molecular targets for the development of agents in the treatment of these diseases. We have previously demonstrated that the gene encoding the hematopoietic-specific truncated isoform of protein tyrosine phosphatase receptor-type O (PTPROt), a phosphatase demonstrating growth suppressor characteristics, is suppressed in chronic lymphocytic leukemia (Clin Cancer Res. 2007 Jun 1;13(11):3174-81). We have further shown that expression of PTPROt is severely impeded by T-cell leukemia 1 (TCL1), an oncoprotein aberrantly expressed in CLL B-cells (Blood. 2011 Oct 14. [Epub ahead of print]). Additionally, PTPROt plays an important role in regulating B-cell receptor (BCR) signaling (J Cell Biochem. 2010 Jul 1;110(4):846-56; Blood. 2006 Nov 15;108(10):3428-33). To comprehend the physiological relevance of PTPROt we have now generated transgenic (Tg) mice expressing PTPROt in B-cells. These mice live their normal life span and exhibit normal B- and T-cell development. Lyn and SYK kinases, the established substrates of PTPROt, are hypo-phosphorylated in B-lymphocytes from the spleen of PTPROt Tg mice relative to those from NTg mice. Because expression of PTPROt is significantly lower in splenic B-lymphocytes from TCL1 Tg mouse model of CLL relative to splenic B-cells from non-transgenic (NTg) mice (Blood. 2011 Oct 14. [Epub ahead of print]), we crossed the TCL1 Tg and PTPROt Tg mice to determine whether expression of PTPROt will inhibit/delay TCL1-mediated leukemogenesis and utilize this model to elucidate the mechanism of PTPROt function in maintaining normal cell physiology. Preliminary gene expression analysis performed on splenic B-cells from NTg, TCL1 Tg and PTPROt/TCL1 double Tg mice demonstrated that the key positive regulators of cell cycle elevated in the TCL1 Tg mice (relative to the NTg mice) are downregulated in the PTPROt/TCL1 double Tg mice (relative to the TCL1 Tg mice). Further, the spleen size as well as the total number of spleen cells was significantly lower in the PTPROt/TCL1 double Tg group relative to the TCL1 Tg group. We are currently in the process of assessing the function of the altered genes in the TCL1 Tg mouse model as well as in human CLL. [Supported by grant CA101956] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1351. doi:1538-7445.AM2012-1351