Graduation Year

2020

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Gary W. Reuther, Ph.D.

Committee Member

Daniel Abate-Daga, Ph.D.

Committee Member

Amer Beg, Ph.D.

Committee Member

Shari Pilon-Thomas, Ph.D.

Committee Member

Pearlie K. Epling-Burnette, Ph.D.

Committee Member

H. Leighton Grimes, Ph.D.

Keywords

cereblon, E3 ubiquitin ligase receptor, functional conservation, hematopoietic stem cell regulation

Abstract

The discovery and implementation of immunomodulatory drugs (IMiD®s) has revolutionized the treatment of many hematological malignancies due to the plethora of IMiD®-induced clinical responses that include anti-angiogenesis, anti-inflammation, and anti-tumor effects, as well as enhanced erythropoiesis, immune modulation and improved metabolism. More ground-breaking was the identification of cereblon as the target of IMiD®s. Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction of neo-substrates by engaging the DDB1-CUL4A-Roc1-RBX1 E3-ubiquitin ligase in human cells but not in mouse cells suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN’s activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN’s non-primate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants of different species retain their drug binding properties to both classical immunomodulatory drugs and to dBET1, a chemical compound and targeting ligand (i.e. a PROTAC) designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN’s E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight provides evidence that mouse CRBN is functional and provides a new avenue of exploring CRBN’s endogenous substrate recruiting function in hematopoietic cells.

Hematopoietic stem cell (HSC) ontogeny is governed by extrinsic and intrinsic programs to control proliferation, localization, differentiation and self-renewal. The E3 ubiquitin ligase substrate receptor CRBN is expressed in hematopoietic lineages, including long-term HSCs (CD48-CD150+Lineage-Sca-1+c-Kit+ (LSK)), but plays an undefined role in hematopoiesis. As CRBN is the only confirmed target of IMiD®s and since IMiD®s have substantial clinical activity in a number of hematological malignancies, CRBN’s involvement in hematopoiesis was investigated. In these diseases, the IMiD®-CRBN interaction is associated with tumor cell death and other cellular responses through at least two major proposed mechanisms including, 1) drug-induced activation of neomorphic protein degradation, and 2) inhibition of endogenous substrate recruitment leading to toxic protein accumulation[3]. To gain insights into the role of CRBN in hematopoiesis, we assessed adult steady state and transplant-associated hematopoiesis and evaluated HSC development during embryogenesis using germline Crbn-/- mice. Compared to Crbn+/+ mice, CRBN deficiency was associated with a 2-3-fold reduction in stem cells in adult bone marrow and a similar reduction in HSCs in the fetal liver at embryonic stage 14.5 when the HSC pool is initiated from several anatomical sites including the yolk sac, aorta gonad mesonephros and fetal liver. Postnatal bone marrow HSCs are maintained throughout life by a process of self-renewal and differentiation. Interestingly, mature lymphoid and myeloid progeny were moderately expanded in peripheral blood of Crbn-/- mice under homeostatic conditions and following serial bone marrow transplantation suggesting that CRBN is critical for stem cell maintenance. RNA-seq analyses and qRT-PCR validation of BM-derived Crbn-/- LSKs and HSCs showed a marked suppression in a “stemness”-related gene signature consisting of Tie2, Hif1α, and Notch1 suggesting that CRBN is required to maintain HSCs in the undifferentiated state. Moreover, high amounts of proliferation within the LT-HSCs at steady-state, increased mature cells at steady-state and following serial transplantation, and increased lethality to repeated 5-fluorouracil (5-FU) challenge in Crbn-/- mice indicate that CRBN maintains HSC quiescence. Collectively our data suggest CRBN functions to maintain HSC quiescence required to maintain the undifferentiated properties of the HSC cells in the adult bone marrow. HSCs reside in highly specialized bone marrow niches where multiple cooperative networks of intrinsic and extrinsic factors are required for HSC homeostasis. Transplantation of Crbn+/+ HSCs into Crbn-/- recipients failed to generate a defined phenotype. However, HSC adhesion (Vcam1, Cxcr4)-associated genes were significantly suppressed. Particularly, the G protein-coupled chemokine receptor CXCR4-CXCL12 interaction which controls fetal and mature BM colonization and regulates homeostatic and regenerative HSC maintenance. These results indicate that CRBN is a functional protein in rodents and demonstrates a novel role for CRBN in the control of hematopoiesis with important implications in understanding how IMiD®s exert their therapeutic actions in hematological cancers.

Our results demonstrate a novel role for CRBN in the control of hemopoiesis with important implications in understanding how IMiD®s exert their therapeutic actions in hematological cancers.

Download

Share

COinS