Rewiring Cancer Drivers to Induce Apoptosis By Transcriptional Chemical Inducers of Proximity in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL), one of the most common types of leukemia among adults in the US, is characterized by the accumulation of CD5+CD19+ monoclonal B cells. Current clinical treatment approaches for progressive CLL patients focus on novel agents targeting Bruton Tyrosine Kinase (BTK) and Bcl-2. Although clinical trials combining novel agent have shown significant efficacy with high overall response and, in some cases, achievement of high rates of undetectable measurable residual disease (MRD), CLL remains incurable with a majority of patients eventually experiencing relapse of their disease. Therefore, it remains of high interest to expand therapeutic options.

Transcriptional chemical inducers of proximity (TCIPs) are small molecules designed and developed by Dr. Gerald R. Crabtree at Stanford University. TCIPs are a new class of anticancer drugs that recruit endogenous cancer drivers or a downstream transcription factor to the promoters of cell death genes and activate their expression, resulting in apoptosis of cancer cells. Current TCIPs are produced by covalently linking small molecules that bind B-cell lymphoma 6 (BCL6) to those that bind to transcriptional activators, such as Bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinases 9 (CDK9). The Stanford team has demonstrated TCIPs generate remarkable killing in diffuse large B cell lymphoma (DLBCL) cell lines, including chemotherapy-resistant, TP53-mutant lines, with a half maximal effective concentration (EC50) of 1-10nM at 72hours (h) and exhibited cell-specific and tissue-specific effects (Gourisankar et al, 2023).

We have initiated TCIPs cytotoxicity assessment in primary leukemic cells from untreated CLL patients. Testing two types of TCIPs with a flow cytometry-based apoptosis assay, we found time and dose-dependent drug-associated killing in primary CLL B cells collected from untreated CLL patients over a 24-72h interval. The IC50 for BRD4-BCL6 TCIP JWZ-7-133 at 72h was 560nM while CDK9-BCL6 TCIP BAK-04-232 at 72h was 10nM. After 24, 48, and 72h exposure of CLL B cells to TCIP JWZ-7-133, the IC50 was 1050, 820 and 560nM, respectively. We also found that TCIP JWZ-7-133 exhibited a similar ability to inhibit leukemic cell viability with an IC50 of 1070nM using a cell viability assay, compared to a flow cytometry-based cell death assay (24h IC50 1050nM).

CDK9-BCL6 TCIPs are designed and produced by covalently linking BI-3812 (binding to BCL6) and SNS-032 (binding to CDK9) with a rigid linker. BI-3812 is a potent and efficacious BCL6 inhibitor, inhibiting the BTB domain of BCL6, and SNS-032 is a potent and selective inhibitor of CDK9. When CLL B cells were treated with only BI-3812 or SNS-032 or TCIP BAK-04-232 as single agents for 72h, we found that TCIP BAK-04-232 had superior and significant killing effects compared to the single agents, with an IC50 of 10nM. Specifically this killing activity was more than 10-fold compared to SNS-032 alone (IC50 over 100nM), and more than 1,000-fold compared to BI-3812 alone (IC50 over 10,000nM).

There remains an urgent to identify new categories of potent drug, inhibitor, or small molecules to expand potential clinical treatment options for patients with CLL. The results presented here indicate that TCIPs targeting BCL6 and BRD4/CDK9 have a strong capacity to induce high levels of cell death in CLL B cells at nanomolar concentrations, and this remarkable killing effect may be due to its unique gain-of-function drug mechanism. TCIPs provide a completely new drug direction and strategy for the design of anticancer drugs: to rewire cancer drivers to activate apoptosis in cancer cells. This study is the first of a kind demonstration of TCIPs in robustly targeting CLL B cells. Further studies to fully validate the preclinical activity of TCIPs and investigate the molecular mechanism(s) for TCIP mediated killing of leukemic B cells will help to define the role of TCIPs as a novel therapeutic approach in CLL.

Han:Recursion Pharmaceuticals: Current equity holder in publicly-traded company. Shanafelt:AbbVie: Research Funding; Pharmacyclics LLC, an AbbVie company: Research Funding; Genentech: Research Funding. Parikh:Amgen: Consultancy; Janssen: Consultancy, Research Funding; BeiGene: Consultancy; Genentech: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Kite: Consultancy; MingSight: Consultancy; Pharmacyclics: Consultancy; AbbVie: Consultancy; Merck: Consultancy, Research Funding; Novalgen Limited: Consultancy. Gray:Matchpoint: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Shenandoah: Current equity holder in private company, Other: Founder, Science Advisory Board Member, Board member; GSK: Current equity holder in private company, Other: Founder, Science Advisory Board Member; C4: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Syros: Current equity holder in private company, Other: Founder, Science Advisory Board Member; CobroVentures: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Soltego: Current equity holder in private company, Other: Founder, Science Advisory Board Member, Board member; Allorion: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Voronoi: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Larkspur: Current equity holder in private company, Other: Founder, Science Advisory Board Member, Board member; Inception: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Lighthorse: Current equity holder in private company, Other: Founder, Science Advisory Board Member; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Jansen: Research Funding; Kinogen: Research Funding; Arbella: Research Funding; Deerfield: Research Funding; Springworks: Research Funding; Interline: Research Funding; Sanofi: Research Funding. Crabtree:Shenandoah Therapeutics: Other: Founder and scientific advisor; Foghorn Therapeutics: Other: Founder and scientific advisor. Kay:Agios Pharma: Other: data safety monitoring committee; Pharmacyclics LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Bristol Meyer Squibb: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Sunesis: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: data safety monitoring committee and advisory board; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Other: data safety monitoring committee and advisory board; BeiGene: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Behring: Membership on an entity's Board of Directors or advisory committees; BMS -Celgene: Other: data safety monitoring committee; Dren Bio: Other: data safety monitoring committee; Vincerx: Research Funding.