Sarott, Roman C.

Abstract Chronic lymphocytic leukemia (CLL) is one of the most common types of leukemia among adults in the US. Despite significant improvement of current novel agent treatments, CLL remains incurable with many eventually experiencing disease relapse. Therefore, it remains of high interest to expand therapeutic options. Transcriptional chemical inducers of proximity (TCIPs) are a new class of anticancer drugs designed and developed by Stanford teams. These agents recruit cancer drivers or transcription factors to activate proapoptotic genes' expression and kill 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) (BRD4-BCL6, TCIP1) or cyclin-dependent kinases 9 (CDK9) (CDK9-BCL6, TCIP2). In collaboration with the Stanford teams, we have initiated TCIPs cytotoxicity assessment in primary leukemic cells from untreated CLL patients and found heterogeneous but evident drug responsiveness with a panel of TCIPs. Our current work has shown that TCIP2 demonstrated remarkable killing ability of CLL B cells with IC50 of 9nM in 72-hours (h), while the IC50 of TCIP1 was 560nM. TCIPs showed time and dose-dependent drug-associated killing in primary CLL B cells over a 24-72h interval using a flow cytometry-based apoptosis assay. After 24, 48, and 72h exposure of CLL B cells to TCIPs, the IC50 was 1054, 820 and 555nM for TCIP1, and 9, 8, and 9nM for TCIP2, respectively. TCIP2 is designed and produced by covalently linking BI-3812 (BCL6 inhibitor) as a BCL6 binder and SNS-032 (CDK9 inhibitor) as a CDK9 binder with a rigid linker. Comparing TCIP2 to its negative controls (TCIP compounds with only one active binder, either BCL6 or CDK9), TCIP2 exhibited significant cytotoxicity in primary CLL B cells. TCIP2 also showed a significant increase in potency, compared to SNS-032 and BI-3812 alone at 72h. Normal human peripheral blood mononuclear cells (PBMCs) were 6-fold less sensitive to TCIP2 compared to CLL PBMCs with high percentage of CLL B cells. Immunoblotting data showed induction of BCL6 protein level after 20nM TCIP1 treatment in CLL B cells. In samples from relapsed/refractory (RR) CLL patients, TCIPs maintained good cytotoxicity with IC50 of 1805nM for TCIP1 and 84nM for TCIP2 in 24h using a cell viability assay. Our study indicates that TCIPs targeting BCL6 and BRD4/CDK9 have a strong capacity to induce high levels of cell death in primary CLL B cells. This notable killing effect may be due to its unique gain-of-function drug mechanism where BCL6 is induced. This is the first study of TCIPs ability to target CLL B cells from both untreated and RR patients. Further studies to more fully evaluate the preclinical activity of TCIPs and investigate the molecular mechanism(s) for TCIP mediated killing of leukemic B cells will help to define the potential role of TCIPs as a novel therapeutic approach in CLL. Citation Format: Weiguo Han, Aisharya Bhattacharya, Basel Karim, Wenzhi Ji, Roman C. Sarott, Meredith Nix, Sai Gourisankar, Andrey Krokhotin, Sutapa Sinha, Zhiquan Wang, Tait D. Shanafelt, Sameer A. Parikh, Nathanael S. Gray, Gerald R. Crabtree, Neil E. Kay. Rewiring cancer drivers to induce apoptosis in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5633.

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.