Date of Award

4-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Marcus Fischer, PhD

Committee

Taosheng Chen, PhD Linda Hendershot, PhD Richard Lee, PhD Wei Li, PhD Bernd Meibhom, PhD Zoran Rankovic, PhD

Keywords

Acute Leukemia, GSPT1/eRF3a, Molecular Glue, Targeted Protein Degradation

Abstract

Acute Lymphoblastic Leukemia (ALL), the most common childhood cancer and the second most common acute leukemia in adults, arises from clonal expansion of undifferentiated lymphoid precursor cells in bone marrow. Despite the extensive knowledge on its cytogenetic and molecular biology, ALL treatment remains highly challenging especially after relapse. Conventional chemotherapy has shown significant improvement in overall survival rates of pediatric patients up to 90%, however, treatment failure due to ALL relapse occurs in 15-20% of the cases. On the other hand, adults and elderly patients with ALL are considered difficult to treat populations with the 5-year overall survival of 30–40%. Small molecule induced protein degradation is a novel strategy that can be applied to currently undruggable targets and oncoproteins. In this paradigm, small molecule degraders (either Proteolysis Targeting Chimera (PROTAC) or Molecular Glue (MG)) redirect the cell’s endogenous ubiquitin proteasome system and induce ubiquitination of the target protein or non-native substrate of an E3 ligase (neosubstrate) and its subsequent proteasomal degradation. Recently, the Cereblon (CRBN) E3 ligase modulator, CC90009 was reported to show potent anti-tumor activity in acute myeloid leukemia (AML), leading to the identification of GSPT1 (G1 to S phase transition factor) as a CRBN neosubstrate. These findings suggest the potential of MGs to target unanticipated vulnerabilities in different malignancies. Here, using a structurally diverse and unique set of MGs (Molecular Glue Library (MGL)) with confirmed CRBN binding affinity, we sought to identify novel CRBN modulators through phenotypic and proteomic methods. Our screening of the MGL in a panel of representative acute leukemia cell lines, including the CRLF2 rearranged ALL cell line MHH-CALL-4 identified several active MGs with EC50 <5 µM. A lenalidomide competition assay and MHH-CALL-4 CRBN knock-down cells confirmed the CRBN dependency of these MGs. Among these compounds, SJ6986, a thalidomide-driven sulfonamide showed potent cytotoxicity in over 10 ALL cell lines tested in vitro. TMT-MS proteomic analyses identified GSPT1/2 as the primary targets for this compound with high selectivity. We next tested SJ6986 activity in a panel of patient derived ALL xenografts (PDX) harboring rearrangement of IGH-CRLF2, EPOR, ATF7IP-JAK2 ex vivo. All the tested tumors were highly sensitive to SJ6986 with IC50 at nanomolar range. PK analyses in mice indicated rapid absorption and over 80% oral bioavailability for SJ6986. PD studies in an IGH CRLF2 PDX showed dose-dependent degradation of GSPT1 within 48 hours of treatment. Finally, we examined the anti-tumor activity of SJ6986 in 6 different PDX representative of high-risk subtypes of ALL including near haploid, low hypodiploid, CRLF2-rearranged and EPOR-rearranged, in vivo for 28 days. SJ6986 was able to dramatically decrease the tumor burden at 1 mg/kg dose in most of the tumor models. Collectively, these results affirm that SJ6986 is a novel CRBN modulator and a potential therapeutic agent by targeting GSPT1 protein with high selectivity and potency for the treatment of ALL.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-8151-6652

DOI

10.21007/etd.cghs.2022.0592

Additional Files
2022-007-Keramatnia-DOA.pdf (148 kB)
Declaration of Authorship
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