Gayathri Natarajan

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.

Ibrutinib enhances IL-17 response by modulating the function of bone marrow derived dendritic cells.

Ibrutinib (PCI-32765) is an irreversible dual Btk/Itk inhibitor shown to be effective in treating several B cell malignancies. However, limited studies have been conducted to study the effect of this drug on myeloid cell function. Hence, we studied the effect of ibrutinib treatment on TLR-4 mediated activation of bone marrow derived dendritic cell culture (DCs). Upon ibrutinib treatment, LPS-treated DCs displayed lower synthesis of TNF-α and nitric oxide (NO) and higher induction of IL-6, TGF-β, IL-10 and IL-18. While ibrutinib dampened MHC-II and CD86 expression on DCs, CD80 expression was upregulated. Further, ibrutinib-treated DCs promoted T cell proliferation and enhanced IL-17 production upon co-culture with nylon wool enriched T cells. Taken together, our results indicate that ibrutinib modulates TLR-4 mediated DC activation to promote an IL-17 response. We describe a novel mode of action for ibrutinib on DCs which should be explored to treat other forms of cancer besides B cell malignancies.

A Tec kinase BTK inhibitor ibrutinib promotes maturation and activation of dendritic cells

ABSTRACT Ibrutinib, a BTK inhibitor, is currently used to treat various hematological malignancies. We evaluated whether ibrutinib treatment during development of murine bone marrow-derived dendritic cells (DCs) modulates their maturation and activation. Ibrutinib treatment increased the proportion of CD11c+ DCs, upregulated the expression of MHC-II and CD80 and downregulated Ly6C expression by DCs. Additionally, ibrutinib treatment led to an increase in MHC-II+, CD80+ and CCR7+ DCs but a decrease in CD86+ DCs upon LPS stimulation. LPS/ibrutinib-treated DCs displayed increased IFNβ and IL-10 synthesis and decreased IL-6, IL-12 and NO production compared to DCs stimulated with LPS alone. Finally, LPS/ibrutinib-treated DCs promoted higher rates of CD4+ T cell proliferation and cytokine production compared to LPS only stimulated DCs. Taken together, our results indicate that ibrutinib enhances the maturation and activation of DCs to promote CD4+ T cell activation which could be exploited for the development of DC-based cancer therapies.

Mechanisms of Immunopathology of Leishmaniasis

While cutaneous leishmaniasis is characterized by lesions proximal to the site of the sandfly bite, visceral leishmaniasis is associated with immunopathology in the liver and spleen of the infected host. This chapter offers a brief overview of the immune responses generated during infections with cutaneous and visceral leishmaniasis. Insights on how the immune responses generated during cutaneous and visceral leishmaniasis coupled with other host- and pathogen-derived factors determine the clinical pathology and outcome of the disease are also discussed.

Subverting Immunity from the Inside: Strategies of Intracellular Survival – Protozoans

Infections caused by intracellular protozoa Leishmania, Trypanosoma, Toxoplasma and, Plasmodium represent a global health challenge. These diseases are responsible for a significant morbidity and mortality worldwide, especially in developing countries. These pathogens usually remain for long periods in their host and sometimes they cannot be eliminated by the immune system or drug treatment. Another important factor that limits the eradication of these pathogens is the lack of a preventive vaccine. This complex scenario is the result of the ability of the parasites to regulate and survive the immune response. One of the more successful strategies developed by these parasites is the ability to infect mammalian cells including those that are designed to fight against infections. Living inside the cell has the advantage to be protected from the immune attack, for example, avoiding the humoral immunity. But, how parasites are able to survive into the cells that defend us from infections? In this article we give a brief overview of the strategies utilized by these parasites to survive inside the host cells.

Transgenic T cell-specific expression of CXCR3 enhances splenic and hepatic T cell accumulation but does not affect the outcome of visceral leishmaniasis

The outcome of visceral leishmaniasis, caused by parasite Leishmania donovani, depends on the recruitment of leishmanicidal Th1 cells. Chemokine receptor CXCR3, preferentially expressed by Th1 cells, is critical for migration of these T cells during infection. During chronic VL, there is a decrease in the presence of CXCR3-expressing CD4+ T cells in the spleen, which is associated with high parasitic burden in this organ. We therefore examined whether T cell-specific expression of CXCR3 in mice (CXCR3Tg) would promote resistance to VL. L. donovani infected CXCR3Tg mice showed increased accumulation of T cells in the spleens compared to WT littermates (CXCR3+/+). However, CXCR3+ T cells from CXCR3Tg mice showed low CD69 expression and these mice developed fewer granulomas. Additionally, both groups of mice showed similar cytokine profiles and parasitic burdens during the course of infection. In summary, although T cell-specific expression of CXCR3 promoted the accumulation of CXCR3-expressing T cells during L. donovani infection, this did not enhance resistance to VL.

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