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3587 Genome-Wide CRISPR/Cas9 Knockout Screen Reveals Novel LP-168 (Rocbrutinib) Combinations Targeting BCL-2 Protein Members for Chronic Lymphocytic Leukemia

Program: Oral and Poster Abstracts
Session: 802. Chemical Biology and Experimental Therapeutics: Poster II
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, Combination therapy, Translational Research, CLL, Diseases, Treatment Considerations, Lymphoid Malignancies
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Britten K. Gordon, BS1, Ethan C. Whipp, MSc2*, Lianbo Yu, PhD1*, Samon Benrashid, BS1*, Andrew D. Mitchell, MS1*, Alexander He, BS1*, Jazmin Urrutia, BS1*, Shrilekha Misra, PhD1*, Alexander R. Marr, BS1, Tzung-Huei Lai, DVM, PhD1*, Naina Karnati1*, Shanmugapriya Thangavadivel, PhD3*, Kerry A. Rogers, MD4, Seema A. Bhat, MD5*, Nicole R. Grieselhuber, MD, PhD6, Yi Chen, PhD7*, Fenlai Tan, MD, PhD8, Stephen P. Anthony, DO9, Yu Chen, MD, PhD7, Deepa Sampath, PhD10, Rosa Lapalombella, PhD1 and Jennifer A. Woyach, MD11

1The Ohio State University, Columbus, OH
2Comprehensive Cancer Center - Wexner Medical Center, The Ohio State University, Columbus, OH
3Division of Hematology, The Ohio State University, Dublin, OH
4Division of Hematology, The Ohio State University, Columbus, OH
5Division of Hematolopgy, The Ohio State University, Columbus, OH
6Division of Hematology, Department of Internal Medicine, The Ohio State University Wexner Medical Center, Columbus, OH
7Newave Pharmaceutical Inc., Pleasanton, CA
8Guangzhou Lupeng Pharmaceutical Co. Ltd, Guangzhou, China
9Newave Pharmaceutical Inc., Colbert, WA
10Department of Hematopoietic Biology and Malignancy, The University of Texas MD Anderson Cancer Center, Houston, TX
11Division of Hematology, Ohio State University Comprehensive Cancer Center, Columbus, OH

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with therapies targeting Bruton’s tyrosine kinase (BTK) and BCL-2, but are limited due to the development of resistance. Dual targeting of BTK and BCL-2 has been efficacious in the clinic, with many patients achieving uMRD and prolonged remission off therapy. We have previously demonstrated the pre-clinical efficacy of LP-168 (Rocbrutinib), a novel selective 4th generation BTKi with an active warhead capable of covalent interaction with WT and T474I BTK and non-covalent binding when a BTK C481 mutation is present (Gordon et al. 2023). This allows for continued BTK inhibition despite development of common resistance mutations. To explore potential combination strategies with LP-168, we utilized a genome wide CRISPR/Cas9 knockout screen with validation of identified targets.

Methods: Genome wide CRISPR/Cas9 knockout screening was performed in HG-3 CLL cells using the Brunello library with analysis via the MAGeCK pipeline. Cells were treated with IC20 (1 uM) of LP-168 for 3 days. Following exclusion of essential genes, negatively selected sgRNAs that were differentially expressed in the LP-168 group were used for analysis. Validation of hits was performed via pharmacological inhibition utilizing CellTiter-Glo, Annexin V/PI, and western blots using HG-3, OSU-CLL, TMD8 WT BTK, TMD8 C481S BTK, and TMD8 T474I BTK cells in addition to primary patient samples. Experiments using TMD8 cells were performed following CRISPR modification to insert either C481S or T474I BTK.

Results: The CRISPR/Cas9 screen revealed 1875 genes with at least 3 out of the 4 sgRNAs depleted in both replicates of the LP-168 treated group vs control. KEGG/GO analysis demonstrated significant enrichment of regulation of mitochondrial membrane potential (p=0.00195) and reactive oxygen species pathways (p=0.00195). As BCL-2 interacts with both pathways, and was amongst the top-ranking genes depleted, we chose to target it with either venetoclax (BCL-2i) or with LP-118, a dual BCL-2/-xL inhibitor currently under investigation for R/R hematological malignancies.

Pharmacologic inhibition of BTK + BCL-2, with either venetoclax (ven) or LP-118, showed synergistic reductions in proliferation of HG-3 (p<0.05, n=3; p<0.05, n=3, respectively), OSU-CLL (p<0.001, n=3, p<0.0001, n=3, respectively), and TMD8 WT BTK (p<0.05, n=3; p<0.01, n=3, respectively) cells at physiologically achievable concentrations (10 nM – 1 uM LP-168 and 10 pM – 10 nM ven or LP-118). LP-168 + ven or LP-118 induced significant cytotoxic effects in OSU-CLL (11%, p=0.001, n=3; 21%, p<0.00001, n=3, respectively) and TMD8 WT BTK (36.4%, p<0.00001, n=3; 66.7%, p<0.00001, n=3, respectively) cells following 72-hour drugging. Next, we utilized primary CLL B cells from treatment-naïve patients and found after both a 24- or 48-hour exposure, the combination of 1 uM LP-168 + 4 nM LP-118 (88%, p<0.00001, n=8) or 1 uM LP-168 + 4 nM ven (73%, p<0.00001, n=8) was able to induce cytotoxicity. Following a 2-hour drugging, LP-168 inhibited both BCR and AKT/mTOR signaling in primary CLL B-cells shown by reduced phosphorylation of BTK, ERK, AKT, and GSK3β, with both combination treatments inducing further inhibition of these targets (98%, p<0.0001, n=4; 49%, p<0.05, n=4; 37%, p<0.05, n=4; 46%, p<0.01, n=4, respectively). Further, we observed retained synergistic effect with both covalent (C481S; p<0.01, n=3) and non-covalent (T474I; p<0.001, n=3) BTKi resistance mutations at physiologically relevant concentrations. Finally, to determine if these combinations retain efficacy in both BTKi and venetoclax resistant settings of CLL, we utilized patient samples who were resistant to both BTKi and ven and treated them with combination of LP-168 + ven and found both combinations retained cytotoxic effect following 24- or 48-hour exposure.

Conclusions: These data show combined use of LP-168 with pharmacological inhibitors targeting BCL-2 and BCL-xL display synergistic activity in CLL, even in the presence of mutations that mediate resistance to BTKi and BCL-2i. These data are consistent with clinical data showing dual targeting of BTK, with previous generation inhibitors, and BCL-2 has been effective in the clinic, with many patients achieving uMRD and prolonged remission off therapy and supports continued preclinical and future clinical investigation of LP-168 with inhibitors of BCL-2 and BCL-2/-xL.

Disclosures: Rogers: Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees; AbbVie Inc, Genentech, a member of the Roche Group, Novartis: Research Funding; AbbVie Inc, BeiGene Ltd, Genentech, a member of the Roche Group, Janssen Biotech Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company, Pharmacyclics LLC, an AbbVie Company: Consultancy; AstraZeneca Pharmaceuticals LP.: Membership on an entity's Board of Directors or advisory committees. Bhat: Abbvie: Consultancy; AstraZeneca: Consultancy, Research Funding; Aptitude Health: Honoraria. Chen: Newave Pharmaceutical Inc.: Current Employment, Current holder of stock options in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Newave Pharmaceutical Inc. Tan: Guangzhou Lupeng Pharmaceutical Co: Current Employment, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Anthony: Sumitomo Dainippon Pharma Oncology: Patents & Royalties; Halia Therapeutics: Consultancy; Exact Sciences: Consultancy; Newave Pharmaceutical Inc.: Current Employment, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Chen: Newave Pharmaceutical Inc.: Current Employment, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Woyach: Newave: Consultancy; Loxo Lilly: Consultancy; Merck: Consultancy; AstraZeneca: Consultancy; Janssen: Research Funding; Genentech, Inc.: Consultancy; BeiGene: Consultancy; AbbVie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Schrodinger: Research Funding; Morphosys: Research Funding.

*signifies non-member of ASH