Abstract The in-clinic phosphatidylinositol 3-kinase (PI3K) inhibitors idelalisib (CAL-101) and duvelisib (IPI-145) have demonstrated high rates of response and progression-free survival in clinical trials of B-cell malignancies, such as chronic lymphocytic leukemia (CLL). However, a high incidence of adverse events has led to frequent discontinuations, limiting the clinical development of these inhibitors. By contrast, the dual PI3Kδ/casein kinase-1-ε (CK1ε) inhibitor umbralisib (TGR-1202) also shows high rates of response in clinical trials but has an improved safety profile with fewer severe adverse events. Toxicities typical of this class of PI3K inhibitors are largely thought to be immune mediated, but they are poorly characterized. Here, we report the effects of idelalisib, duvelisib, and umbralisib on regulatory T cells (Tregs) on normal human T cells, T cells from CLL patients, and T cells in an Eμ-TCL1 adoptive transfer mouse CLL model. Ex vivo studies revealed differential effects of these PI3K inhibitors; only umbralisib treatment sustained normal and CLL-associated FoxP3+ human Tregs. Further, although all 3 inhibitors exhibit antitumor efficacy in the Eμ-TCL1 CLL model, idelalisib- or duvelisib-treated mice displayed increased immune-mediated toxicities, impaired function, and reduced numbers of Tregs, whereas Treg number and function were preserved in umbralisib-treated CLL-bearing mice. Finally, our studies demonstrate that inhibition of CK1ε can improve CLL Treg number and function. Interestingly, CK1ε inhibition mitigated impairment of CLL Tregs by PI3K inhibitors in combination treatment. These results suggest that the improved safety profile of umbralisib is due to its role as a dual PI3Kδ/CK1ε inhibitor that preserves Treg number and function.

Although the treatment paradigm for chronic lymphocytic leukemia (CLL) is rapidly changing, the disease remains incurable, except with allogeneic bone marrow transplantation, and resistance, relapsed disease, and partial responses persist as significant challenges. Recent studies have uncovered roles for epigenetic modification in the regulation of mechanisms contributing to malignant progression of CLL B cells. However, the extent to which epigenetic modifiers can be targeted for therapeutic benefit in CLL patients remains poorly explored. We report for the first time that expression of epigenetic modifier histone deacetylase 6 (HDAC6) is upregulated in CLL patient samples, cell lines, and euTCL1 transgenic mouse models compared with HDAC6 in normal controls. Genetic silencing of HDAC6 conferred survival benefit in euTCL1 mice. Administration of isoform-specific HDAC6 inhibitor ACY738 in the euTCL1 aging and adoptive transfer models deterred proliferation of CLL B cells, delayed disease onset via disruption of B-cell receptor signaling, and sensitized CLL B cells to apoptosis. Furthermore, coadministration of ACY738 and ibrutinib displayed synergistic cell kill against CLL cell lines and improved overall survival compared with either single agent in vivo. These results demonstrate for the first time the therapeutic efficacy of selective HDAC6 inhibition in preclinical CLL models and suggest a rationale for the clinical development of HDAC6 inhibitors for CLL treatment, either alone or in combination with Bruton tyrosine kinase inhibition.

Background: Basic and clinical scientific research at the University of South Florida (USF) have intersected to support a multi-faceted approach around a common focus on rare iron-related diseases. We proposed a modified version of the National Center for Biotechnology Information’s (NCBI) Hackathon-model to take full advantage of local expertise in building “Iron Hack”, a rare disease-focused hackathon. As the collaborative, problem-solving nature of hackathons tends to attract participants of highly-diverse backgrounds, organizers facilitated a symposium on rare iron-related diseases, specifically porphyrias and Friedreich’s ataxia, pitched at general audiences. Methods: The hackathon was structured to begin each day with presentations by expert clinicians, genetic counselors, researchers focused on molecular and cellular biology, public health/global health, genetics/genomics, computational biology, bioinformatics, biomolecular science, bioengineering, and computer science, as well as guest speakers from the American Porphyria Foundation (APF) and Friedreich’s Ataxia Research Alliance (FARA) to inform participants as to the human impact of these diseases. Results: As a result of this hackathon, we developed resources that are relevant not only to these specific disease-models, but also to other rare diseases and general bioinformatics problems. Within two and a half days, “Iron Hack” participants successfully built collaborative projects to visualize data, build databases, improve rare disease diagnosis, and study rare-disease inheritance. Conclusions: The purpose of this manuscript is to demonstrate the utility of a hackathon model to generate prototypes of generalizable tools for a given disease and train clinicians and data scientists to interact more effectively.

Recently, genome-wide association study reveals a significant association between specific single nucleotide polymorphisms (SNPs) in men and their sexual orientation. These SNPs (rs9547443 and rs1035144) reside in the intergenic region between the SLITRK5 and SLITRK6 genes and in the intronic region of the TSHR gene and might affect functionality of SLITRK5, SLITRK6, and TSHR proteins that are engaged in tight control of key developmental processes, such as neurite outgrowth and modulation, cellular differentiation, and hormonal regulation. SLITRK5 and SLITRK6 are single-pass transmembrane proteins, whereas TSHR is a heptahelical G protein-coupled receptor (GPCR). Mutations in these proteins are associated with various diseases and are linked to phenotypes found at a higher rate in homosexual men. A bioinformatics analysis of SLITRK5, SLITRK6, and TSHR proteins is conducted to look at their structure, protein interaction networks, and propensity for intrinsic disorder. It is assumed that this information might improve understanding of the roles that SLITRK5, SLITRK6, and TSHR play within neuronal and thyroidal tissues and give insight into the phenotypes associated with male homosexuality.

INTRODUCTION: TGR-1202 is a novel, next-generation PI3Kδ inhibitor presenting significant structural and pharmacological differences from prior small-molecule PI3Kδ inhibitors. TGR-1202 has high clinical efficacy in treatment of B cell malignancies with a substantially differentiated adverse event profile compared to previous PI3Kδ inhibitors, specifically concerning hepatotoxicity or colitis which have been minimal or non-existent. It has been postulated that these effects may be due to T cell immune-mediated mechanisms. We hypothesized that TGR-1202 preserves the function of the regulatory T cell (Treg) population, translating to decreased immune-mediated side effects after treatment. Here, we aimed to compare effects of clinically available PI3Kδ inhibitors on T cells with an emphasis on Tregs. METHODS: We compared activity of idelalisib, duvelisib, and TGR-1202 as single agents in vitro in isolated human T cells. Viability, apoptosis, and proliferation were determined using CellTiter Blue®, annexin V/PI and CFSE staining. CBA and qRT PCR were used to measure cytokines and transcription factors. Flow cytometry was used to detect subset ratios and surface marker expression. RESULTS: First, we observed comparable dose-dependent increases in cytotoxicity beginning at 25uM following treatment of isolated T cell populations with idelalisib, duvelisib, or TGR-1202. At this dose, apoptosis was induced between 48 and 72h. Second, all inhibitors reduced cytokine production in CD3+ T cells upon stimulation. Particularly, IFN-γ, IL-10 and IL-17a reduction was less pronounced after TGR-1202 treatment, indicating relative conservation of T cell response. All inhibitors lowered mRNA expression of T-bet (Th1), GATA-3 (Th2) and FoxP3 (Treg), however FoxP3 levels were consistently higher in TGR-1202 treated T cells. Third, we detected normal CD4:CD8 ratio and unaffected proliferative capacity of CD4+ and CD8+ subsets after drug treatment. Finally, all inhibitors decreased total percent of Tregs following stimulation (CD4+ CD25HI FoxP3+) accompanied by decreased expression of co-inhibitory molecules CTLA-4 and PD-1 on the Tregs. Interestingly, TGR-1202 significantly preserved the percent of Tregs closer to normal as well as surface expression of CTLA-4 and PD-1 on Tregs, indicating greater retention of immune checkpoint blockade and suppressive phenotype. CONCLUSIONS: We report herein that TGR-1202 affects human T cells differently than idelalisib and duvelisib. TGR-1202 sustains IL-10 production, FoxP3 mRNA expression, and maintains Treg percentage and expression of immune checkpoint molecules, suggesting relative preservation of numbers and function of Tregs. Data presented begin to provide novel insight into immune mediated cellular mechanisms responsible for lack of side effects in clinical trials of TGR-1202. In vivo models to further characterize effects on the T cell compartment are ongoing. Citation Format: Kamira K. Maharaj, John Powers, Renee Fonseca, Hari Miskin, Dave Maryanski, Eva Sahakian, Javier Pinilla-Ibarz. Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 545.