Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Javier Pinilla-Ibarz, M.D., Ph.D.

Committee Member

Jose Conejo-Garcia, M.D., Ph.D.

Committee Member

Sheng Wei, M.D.

Committee Member

P.K. Epling-Burnette, Ph.D., Pharm.D.

Committee Member

Shari Pilon-Thomas, Ph.D.

Committee Member

John Pagel, M.D.

Keywords

B cell, CLL, HDAC, ibrutinib, immune, T cell

Abstract

Chronic lymphocytic leukemia (CLL) is the most common leukemia in developed countries. It is characterized by the accumulation of CD5+ B lymphocytes in the peripheral blood, bone marrow, and lymphoid tissues of affected patients. Patients experience lymphadenopathy, fatigue, and are severely immunocompromised. Management of disease and symptoms is possible with chemo-immunotherapy for higher risk patients; however, CLL remains incurable unless bone marrow transplant is performed. Identification of novel targets and optimization of current therapeutics is therefore still necessary. The new targeted therapies are at the forefront of development for CLL treatment, but it remains challenging to maximize their efficacies while avoiding toxicities.

In this body of work, experimental results demonstrated a role for epigenetic modifier histone deacetylase 6 (HDAC6) in CLL B-cell biology and suggest that targeting of HDAC6 is an effective, novel strategy against chronic lymphocytic leukemia. Antitumor effects occurred through blockade of intrinsic survival signaling and through beneficial immunomodulation. Briefly, this data showed that genetic silencing or pharmacological inhibition of HDAC6 in preclinical CLL mouse models reduced tumor burden and increased survival through inhibition of pro-survival, anti-apoptotic B-cell receptor signaling in CLL B cells. Further interrogation of B-cell receptor signaling was performed ex vivo, showing transcriptional downregulation of spleen tyrosine kinase (SYK) and reduced protein expression of anti-apoptotic transcription factors myeloid cell leukemia (MCL)-1 and B cell lymphoma (BCL)-2.

Based on the ability of HDAC6 to alter intrinsic survival signaling, a rational combinatorial treatment with ibrutinib, a frontline CLL therapeutic, was tested. The combination of HDAC6 inhibitor plus ibrutinib showed synergistic cell kill in CLL cell lines and patient-derived B cells, reduced tumor burden, and increased survival in the preclinical CLL mouse model. HDAC6 was also found to play a role in immunomodulation of CLL B cells and CLL T cells in the microenvironment. HDAC6 silencing or inhibition modified the expression of immunoreactive molecules on CLL B and T cells, including programmed cell death (PD)-1, programmed cell death ligand (PD-L)-1, major histocompatibility complex (MHC)II and lymphocyte-activation gene (LAG)-3. Interestingly, alteration of STAT3 signaling was involved in the immunomodulatory activity of HDAC6. As a result, HDAC6 inhibition resulted in a beneficial reversal of T-cell dysfunction. Moreover, HDAC6 inhibition augmented the antitumor efficacy of immune checkpoint inhibitor, anti-PD-1.

B-cell receptor (BCR) and phosphoinositol-3-kinase (PI3K) signaling have emerged as therapeutic avenues that have changed the paradigm of CLL treatment. Inhibitors targeting these pathways abrogate survival signaling in B cells, but also can affect other immune compartments. Secondary immune targets result in a variety of physiological effects that are not thoroughly understood. On this note, we investigated the role of CLL T-cell subsets in the incidence of autoimmune-related toxicities occurring in patients treated with PI3K inhibitors idelalisib, duvelisib and umbralisib. The data established the regulatory T-cell subset (Tregs) as a definitive regulator of the autoimmune-like toxicities using preclinical CLL and graft-versus-host models. Finally, side-by-side comparison of clinically available PI3K inhibitors demonstrated that umbralisib exhibits differential and beneficial effects on Tregs through inhibition of casein kinase (CK)-1 epsilon and canonical Wnt signaling.

The work reported here contributes knowledge and furthers understanding of immunomodulatory approaches using small-molecule inhibitors for treatment of CLL. While chemo-immunotherapy is still used and appropriate for selected younger patients, alternative strategies are sought after for the unfit, elderly, resistant, relapsed and refractory patients. Novel therapeutic strategies or rational combinations will be most beneficial to those patients who progress or develop resistance on established regimes. Ultimately, identification of novel targets and optimization of existing therapies is necessary to increase responses, eradicate minimal residual disease and mediate toxicity. These processes will advance the treatment regimen for chronic lymphocytic leukemia patients and impact quality and length of life.

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