Citations

Abstract Epigenetic inheritance relies on the precise recycling of parental histones during DNA replication. The N-terminal region of Pol1, the catalytic subunit of DNA polymerase α, has previously been implicated in chaperoning parental histone recycling. In this study, we identify a single amino acid mutation in the C-terminal region of Saccharomyces cerevisiae Pol1 (pol1-F1396S) that destabilizes the protein and disrupts the transfer of parental histones to the lagging strand during DNA replication. As a primary consequence, pol1-F1396S cells exhibit decreased chromatin stability. Furthermore, we demonstrate that the cellular abundance—or dosage—of all three DNA polymerases (Pol α, Pol δ, and Pol ε) influences the distribution of parental histones between the leading and lagging strands, revealing a dosage-sensitive mechanism of epigenetic regulation during DNA replication.

Abstract Bruton tyrosine kinase (BTK) is central to chronic lymphocytic leukemia (CLL) biology, yet the regulatory programs that depend on BTK remain poorly defined. Here, we used acute NX-5948 (Bexobrutideg)-mediated BTK degradation to define BTK-dependent regulatory circuitry in CLL. Integrating early transcriptional and chromatin responses with chromatin-state annotation, target-gene linkage, and transcription factor assignment, we resolved a chromatin-informed framework linking BTK loss to downstream gene-regulatory output. This framework is separated into two directional programs: a dominant BTK-maintained network that collapsed after BTK degradation and a smaller BTK-released network that emerged upon BTK loss. BTK-responsive elements were concentrated in promoter- and enhancer-linked chromatin compartments, consistent with a model in which BTK-dependent transcriptional output is mediated through specific active regulatory elements. Functional validation in a CLL-like cell model provided orthogonal support for the NFAT-linked component of the BTK-maintained regulatory circuitry. BTK-dependent regulon activity was associated with clinical outcome, and projection of bulk-derived programs into longitudinal single-cell RNA-seq resolved malignant CLL cells into distinct regulatory states with maintained-like and released-like features. Together, this work defines BTK-dependent regulatory programs in CLL through a chromatin-informed framework linking BTK activity to gene-regulatory output. These findings lay the foundation for understanding how BTK-dependent circuitry contributes to malignant cell-state diversity, clinical outcome, and remodeling during therapy.

Abstract Background: CLL incidence differs by ancestry, with higher rates in individuals of European ancestry (EA) than African ancestry (AA). The contribution of epigenetic variation to these disparities remains unclear. We investigated chromatin accessibility, histone modifications, and gene expression in CLL patients of EA and AA to identify ancestry-associated epigenetic signatures. Methods: ATAC-seq was performed on blood clonal B cells from 15 AA and 51 EA patients; CUT&Tag profiling of six histone marks (H3K4me1, H3K4me3, H3K27ac, H3K9me3, H3K27me3, H3K36me3) was done in 8 AA and 12 EA cases; RNA-seq was generated for all samples. Ancestry was genetically confirmed. Differential regions were linked to genes using ENCODE and Hi-C datasets. Results: ATAC-seq revealed 4,955 regions with increased and 3,000 with decreased accessibility in AA vs EA (fold change >2, FDR ≤0.05). Regions with increased accessibility mapped to 2,313 genes; 599 were upregulated and enriched in TNF-α/NF-κB (p=1.40E-53) and apoptosis pathways (p=6.90E-11). Regions with decreased accessibility linked to 1,953 genes; 317 were downregulated, enriched in interferon-γ response (p=1.04E-03) and IL-6/JAK/STAT3 signaling (p=6.41E-03). Histone profiling showed ancestry-specific changes across all marks. For H3K27ac (active enhancer), 68 genes with a decreased signal in AA were downregulated and enriched in interferon-γ (p=1.55E-06) and IL-6/JAK/STAT3 (p=9.62E-05) pathways. Increased H3K27me3 (repressive histone modification) in AA affected 399 genes enriched in IL-6/JAK/STAT3 (p=8.13E-09) and interferon-γ (p=2.48E-05) pathways. These findings suggest coordinated regulation by active and repressive chromatin states. Conclusions: Despite limited sample size, this study suggests differences between AA and EA ancestries may exist in epigenetic signatures in CLL, implicating chromatin accessibility and histone modifications in pathways relevant to tumor biology. These signatures may also contribute to the differences in CLL incidence by ancestry. Future studies with more patients and functional validation experiments are needed to clarify the role of these epigenetic differences in CLL pathogenesis. Citation Format: Zhiquan Wang, Huihuang Yan, Mingma Sherpa, Daniel Bihnam, Cristine Allmer, Chantal McCabe, Daniel O’Brien, Dennis P. Robinson, James R. Cerhan, Neil E. Kay, Sameer A. Parikh, Esteban Braggio, Susan Slager. Ancestry-associated epigenetic differences in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5064.

Abstract Introduction: A subset of patients with chronic lymphocytic leukemia (CLL) experiences disease progression from CLL to Richter transformation (RT), characterized by the occurrence of a high-grade lymphoma. The outcomes of patients with RT are typically dismal and identifying better therapeutic strategies remains critically important for these patients. To discern the biologic underpinnings driving this transformation, we studied immune changes and genetic evolution in samples from CLL patients who developed RT. Methods: We identified 18 patients with CLL who developed RT; and performed single cell (sc) RNA-sequencing(seq) on sorted B and T cells from peripheral blood mononuclear cells (PBMCs) at the time of RT (n=18) and at the earlier time of CLL diagnosis (n=11). The median time between the CLL sample and the one closest to RT was 4.3 years (range, 0.2-7.3); and patients received a median of 1 CLL directed treatment (range, 0 – 7). A total of 749,906 cells and 36,601 genes were captured during scRNAseq and 720,580 cells and 21,892 genes were retained for downstream analysis, respectively. Analysis was performed using the immunopipe (https://github.com/pwwang/immunopipe) that wraps around Seurat (v5.1.0) and identified enriched pathways using Enrichr service. The sc-T cell receptor (TCR)-repertoire(R)-seq data was analyzed and visualized using scplotter (). The whole exome sequencing (WES) analysis with tumor B-cells from same patients of CLL & RT stages were done using the Agilent sureselect Human all exon v5 + UTR capture kit. Reads from paired end sequencing were processed with the sarek pipeline. Results: Analysis of 720,580 cells from 11 CLL and 18 RT samples showed 16 distinct cell types- 3 B and 13 T cell subtypes. We observed an increasing trend of CD8 TEM [mean 0.548 vs. 0.384, p = 0.12 (Wilcoxon test)] but a decreasing trend of CD4 TCM (mean 0.236 vs. 0.337, p = 0.08) and B-memory (mean 0.0085 vs. 0.0195, p = 0.51) subsets in RT compared to their CLL stage. When comparing transcriptional changes, we found that among the top 18 highly variable genes, the expression of FP236383.3 (lncRNA) and TCL1A was significantly higher (p<0.0001) in the B cell subsets of patients with RT vs. CLL stage. Additionally, in T cell subsets—primarily dnT, γδT, and CD8/CD4 naïve cells—the expression of FP236383.3, NKG7, GNLY, and CCL5 was significantly higher (p<0.0001), while the expression of LDHB, GZMK, and IL32 was significantly lower (p<0.0001) in RT vs. CLL stage samples. Comparison of specific gene set enrichment analysis showed significantly increased expression of GZMB in gdT (p<0.001) and decreased expression of GZMA, GZMK, TIGIT and TCF7 in dnT and CD8 Naïve cells (p<0.001) for RT vs. CLL stage. The top 10 differentially expressed pathways in RT included oxidative phosphorylation, allograft rejection, IFN-α/-γ responses, TNF-α signaling via NF-κβ, complement, Myc targets V1, mTORC1, hypoxia, and p53 signaling across various B and T cell subsets compared to the CLL stage. Initial analysis of TCR-R data showed that patients with RT stage have lower number of unique clones (p<0.5) and reduced clonal diversity (p<0.5), and a higher number of large and hyperexpanded clones compared to their CLL stage. Additionally, WES data indicated that the most frequently mutated gene is TP53 in tumor B cells of both CLL and RT stages. However, a higher percentage of RT-stage samples carry mutations in MGA, ATM, and IRF2BP2 genes compared to their CLL-stage. WES data also showed a trend toward differing frequencies of copy number amplification at 19q12 (p=0.10) and 4q13.1 (p=0.52) between RT and CLL stages. Conclusions: The main findings of our study are as follows:The transcriptional landscape showed significant changes in multiple genes and pathway enrichment related to cell growth, differentiation and immune response in RT vs. CLL stage.A decreased fraction of CD4 TCM cells and reduced TCR clonal diversity were observed in samples at the RT stage and may indicate a poor prognosis for the patients at this stage. WES data of tumor B cells also indicated possible genetic alterations in the RT vs. CLL stage. Data from in-depth analyses on understanding of the dynamics of T cell response and additional experiments on these patients are expected to provide valuable insights into tumor genetic heterogeneity, transcriptional and immune regulation in blood-based immune subsets.

Abstract Introduction: Although current clinical treatment approaches targeting Bruton Tyrosine Kinase (BTK) and BCL2, either as monotherapy or in combination are highly effective in chronic lymphocytic leukemia (CLL), CLL remains incurable. Identifying novel drugs that are potent and effective in the relapsed/refractory (RR) setting remains an unmet need. Methods: Utilizing the CellTiter-Glo 2.0 Cell Viability Assay, we have developed a preclinical drug screening platform which allows conducting drug sensitivity profiling of primary peripheral blood mononuclear cells (PBMCs) collected from CLL patients to a panel of drugs, in the presence and/or absence of bone marrow stromal cells (BMSCs). This unique system mimics the in vivo stromal microenvironment in CLL and enables testing of multiple drug responses in primary patient samples, particularly when leukemic cell numbers are low. In this study, we employed this high throughput drug screening platform to profile drug sensitivity in CLL B cells after disease progression on a BTK inhibitor (BTKi) to a panel of 12 drugs, including inhibitors of BCL2 (venetoclax and LP-118 [Newave]), AXL (TP-0903), histone deacetylase (panobinostat and belinostat), p53 (eprenetapopt and idasanutlin), proteasome (carfizomib) and Type III Receptor Tyrosine Kinase (crenolanib), next generation BTKi (LP-168 [Newave]), as well as a novel anti-cancer drug transcriptional chemical inducer of proximity (TCIP1) and the natural green tea extract (GTE) product Sunphenon 90D (Taiyo International). After 24-hour drug exposure, leukemic cell viability was measured, killing curves and IC50 levels generated and calculated by nonlinear curve regression in GraphPad Prism. Results: Using the Mayo Clinic CLL Database and the Mayo Clinic CLL Tissue Bank, we identified 47 patients with RR CLL, all of whom experienced disease progressionon a BTKi (35 ibrutinib +/- CD20 antibody, 10 acalabrutinib +/- CD20 antibody, 2 other). Median age at BTKi initiation was 64 years (range, 41-82), and 36 (77%) were male. IGHV status was unmutated in 44 (94%) patients, FISH at diagnosis showed del17p in 11 (27%), del11q in 6 (15%), +12 in 5 (12%), del13q in 12 (30%), and not detected in 6 (15%) patients. TP53 mutation was seen in 13 (32%) patients at the start of BTKi therapy. Median number of lines of CLL therapy prior to start of BTKi was 2 (range, 0–8); 13 patients received BTKi as their first-line therapy. Our drug screening results showed definite responses to all tested drugs based on dose range generated killing curves and IC50 levels for 30 RR CLL patient samples, though results varied. Among 12 drugs tested, LP-118 (0.1nM), venetoclax (2.6nM), carfizomib (2.2nM), TP-0903 (116.2nM), crenolanib (1769nM), and TCIP1 (1805nM) demonstrated the highest killing effects, while the remaining generated killing but at higher doses: belinostat (6800nM), eprenetapopt (4500nM), idasanutlin (4900nM), LP-168 (7500nM) and GTE (55.6uM). We also observed that RR CLL cohort exhibited significantly increased drug resistance to most of the tested drugs in the presence of BMSCs with corresponding increases in IC50s (with vs. without BMSCs, fold-change respectively): LP-118 3.92-fold, venetoclax 1.98-fold, carfilzomib 2.58-fold, TP-0903 1.65-fold, panobinostat 204.23-fold, TCIP1 1.91-fold, crenolanib 1.31-fold, idasanutlin 1.69-fold, belinostat 6.14-fold, LP-168 1.29-fold, eprenetapopt 3.44-fold and GTE 1.08-fold (11 out of 12 with p-values <0.05 except for GTE which was not significant, Mann-Whitney U test). We found that even for three drugs with the strongest killing effect based on IC50 levels (LP-118, venetoclax, carfilzomib), their killing effects were significantly reduced in the presence of BMSC (p=0.0002, 0.0002, and <0.0001, respectively). Conclusions: Our results highlight the spectrum of currently available drugs that show promise for therapeutic use in patients with RR CLL who experience disease progression on BTKi. The killing impact was still seen, albeit diminished, even in the presence of BMSC. Our ongoing work will be to associate the drug profiles with each patient's clinical history, as well as to the comprehensive genetic and epigenetic features of the CLL B cell clone.

Abstract Introduction: Although therapies targeting Bruton Tyrosine Kinase (BTK), either as monotherapy or in combination have revolutionized the treatment in chronic lymphocytic leukemia (CLL), the disease remains incurable. Continuous treatment with these agents frequently leads to the development of resistance and disease progression, mostly associated with mutations in BTK or PLCG2. However, a significant fraction of BTKi-resistant patients does not harbor mutations in these genes, revealing that multiple distinct mechanisms of resistance can independently develop in these patient cohorts. Identifying novel molecular pathways associated with drug resistance in the relapsed/refractory (RR) setting of CLL requires more exploration. Materials and Methods: Using the Mayo Clinic CLL Database and tissue bank, we identified 30 RR patients, who had had disease progressionon a BTKi. The median lines of prior therapies were four. The median age at start of treatment was 57.9 (range, 36-78), 77% were male. Rai stage at diagnosis was 0 in 10% of patients, stage I in 37%, stage II in 20%, stage III in 6%, and stage IV in 27%. IGHV unmutated was observed in 87% of cases. Five patients subsequently evolved to Richter's Transformation (RT). A total of 64 blood samples were collected at different timepoints: baseline (before BTKi therapy), during treatment and at disease progression from BTKi (Ibrutinib/Acalabrutinib/Pirtobrutinib/Nemtabrutinib). The CD5+/CD19+ tumor population was enriched by flow sorting. We performed whole genome sequencing (WGS) in tumor/germline pairs with an average sequencing depth of 60X. Copy number variants (CNVpytor), structural variants – SV (Manta and DELLY2), single nucleotide variants (SNV) and indels (MuTect2, Strelka) were calculated. Mutational signature analysis was conducted independently on each disease timepoint with sigProfiler. De novo signatures were extracted with non-negative matrix factorization, then decomposed and mapped to known signatures from the COSMIC Human Cancer Signatures database. Results: Prior to start of BTKi treatment, the most common abnormality was del13q (65%), followed by abnormalities in TP53 (mutation and/or del17p) in 59% of patients, del8p, del11q (23% each), and mutations in SF3B1 (35%), NFKIE, NOTCH1 and MAPK signaling pathway (BRAF, KRAS, NRAS, PTPN11) in 23% of cases, each. Complex karyotype, defined as >3 copy number abnormalities was found in 59% of cases. Disease progression was observed 4.4 years (median; range 1.9-10.8 yrs) after starting BTKi therapy. At disease progression, there was an overall increase in genomic complexity. Overall, 17 cases (56%) showed mutations in BTK (p.C481S, p.C481Y, p.L528W, p.A428D) and/or PLCG2 (p.P844H). Four cases showed mutations in both genes or two or more independent mutations in BTK. Del17p and/or TP53 mutations were found in 20 (67%) cases, 9 of them with biallelic abnormalities. A third of patients (N=10) were found to have MAPK mutations, and 9 (30%) cases had mutations in the epigenetic regulators CREBBP, SETD2, SETD1A andKMT2D. Loss of heterozygosity, including 11q and 17p was found in 13% of cases. Chromothripsis was found in 23% of cases. In patients without BTK or PLCG2 mutations (N=13), TP53 abnormalities were present in 92% of cases and mutations in the MAPK pathway in 46% of these patients. Finally, we identified several SVs involving driver genes at disease progression, including inversions in RB1 (N=1), NOTCH1 (N=1) and translocations in MYC (N=1) and IGH (N=2). Analyzing the mutational signatures, baseline timepoints were dominated heavily by aging signatures (SBS5, SBS1). However, in cases that progressed after therapy, we found enrichment of mutational signatures associated with defective DNA mismatch repair (SBS44, 6%), chemotherapy treatment (SBS25, 14%), homologous and nucleotide excision repair deficiency (SBS8, 48%), and indirect effects of AID-induced somatic mutagenesis (SBS85, 7%). Additionally, the Melphalan signature (SBS99, 10%) was found in patients treated alkylating agents. Conclusions: Disease progression of CLL on BTKi confirmed the existence of multiple driver genes beyond BTK, PLCG2 andTP53, including the high prevalence of mutations in MAPK pathway and epigenetic regulators, and increased genomic complexity. Ongoing efforts are focused on integrating these findings with clinical demographics, epigenetic modifications, and preclinical drug sensitivity profiles.

Abstract Twenty percent of patients with follicular lymphoma relapse early with poor outcomes; however, the molecular mechanisms underlying this aggressive behavior are unknown. Using a multiomic approach, we show that patients with follicular lymphoma with elevated IRF4 expression (IRF4hi) have increased transformation risk, dysregulated immune signaling, and a suppressive tumor microenvironment. Loss- and gain-of-function experiments in IRF4hi lymphoma cells, along with chromatin profiling, demonstrate that IRF4 impairs their interaction with T cells by repressing antigen presentation and co-receptor gene modules while promoting the expression of cytokines that antagonize T-follicular helper cell and regulatory T-cell functions. Additionally, IRF4 rewires tumor metabolism and restricts glucose availability to immune cells. Silencing of IRF4 inhibits tumor cell growth and restores immune surveillance mechanisms, thus representing a promising target for therapy. Our data suggest that IRF4hi lymphoma cells co-opt a developmental mechanism used to exit the germinal center response in promoting a more aggressive cancer via engagement of multiple immune-evasive mechanisms. Significance: We show that IRF4 controls immune and metabolic signaling with functional effects on the cross-talk between B and T cells, resulting in a suppressive immune microenvironment when IRF4 is overexpressed in lymphoma. This underscores the importance of IRF4 as a pivotal therapeutic target to restore antitumor immunosurveillance for IRF4-associated lymphomas.

Abstract Most disease-associated single-nucleotide polymorphisms (SNPs) identified by genome-wide association studies (GWASs) reside in noncoding regions. There is increasing evidence that they play regulatory roles through epigenetic mechanisms by altering local chromatin state, 3D chromatin structure, and looping interactions. We and others have identified 42 CLL risk loci from GWASs. At 12 risk loci located outside of gene promoters, we previously demonstrated that functional SNPs are highly enriched in enhancers and super-enhancers that mostly target BCL2 family genes and transcriptional regulators. However, the study was limited by using only public epigenomic data generated in lymphoblastoid cell lines (LCL) and normal immune related cell types. To gain a better understanding of how the functional SNPs alter local chromatin state and modulate target gene expression in CLL, we generated RNA-seq data (n=588) and transposase-accessible chromatin with sequencing (ATAC-seq) data (n=69) in CLL tumor B cells. We performed molecular quantitative trait loci (QTL) analysis with index and correlated SNPs (R2 0.5) at 41 risk loci (excluding the HLA locus). The analyses identified cis expression QTLs (eQTLs), within 1Mb of genes’ transcription start site, at 25 risk loci and chromatin accessibility QTLs (caQTLs, within peak) at 11 risk loci, with both eQTLs and caQTLs at 8 risk loci. We identified a total of 54 eGenes, for which the expression is affected by nearby SNPs within 1Mb, including 40 novel eGenes at 16 CLL risk loci not previously reported. Notably, based on publicly available chromosome interaction data from CLL, LCL, and normal B cells, for over 70% of the novel eGenes, their promoters showed looping interactions with the regions carrying the eQTLs. Further, 13 SNPs were identified as both caQTL and eQTL, with the associated novel eGenes including CD81 at 11p15.5, IPCEF1 at 6q25.2, as well as CXXC1 and MBD1 at 18q21.1. The result suggests that these SNPs play a role in altering chromatin accessibility contributed to gene expression variation. At 11p15.5 risk locus, SNP rs2651823 (A/G) represents a novel caQTL and eQTL. The alternative G allele is associated with increased chromatin accessibility and CD81 expression. Hi-C data from GM12878 and CD20+ B cells supported interaction between the open chromatin region carrying rs2651823 and CD81 promoter. CD81 facilitates the trafficking of CD19 to the surface of activated B cells, and CD81-null mice showed impaired immune response. In a mouse model, CD81 contributes to tumor progression and metastasis in Lewis lung carcinoma. In summary, our molecular QTL analysis revealed that functional SNPs, located in CLL risk loci, modulate chromatin accessibility and expression of downstream target genes that could impact CLL risk. Citation Format: Huihuang Yan, Zhiquan Wang, Mingma Hoel, Cristine Allmer, Chantal McCabe, Daniel O’Brien, Dennis Robinson, James Cerhan, Sameer Parikh, Esteban Braggio, Susan Slager. Molecular QTL analyses identify functional variants at inherited risk loci 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 2763.

Abstract Despite the advent of novel targeted therapies with high overall response rates, chronic lymphocytic leukemia (CLL) is still largely incurable due to the development of resistance. The bone marrow microenvironment (BMME) plays an important role in the survival and drug resistance in CLL. BM stromal cells (BMSCs) are considered a key component of BMME. Our long-term goal is to gain further insights of BMSCs-CLL crosstalk influencing both survival and drug resistance in CLL. To understand the global biologic changes that occur in leukemic CLL cells when in contact with BMSCs, we assessed RNA-seq in paired CLL cells isolated from untreated blood and BM and in untreated patients’ CLL cells cultured alone or with BMSCs. CLL cells from blood/BM and cultured alone/with BMSCs were also examined by Immunoprecipitation (IP), Western blot (WB), untargeted metabolomics (LCMS+GCMS), and preclinical drug sensitivity assays. We detected only 13 upregulated genes that overlap between CLL cells from BM and cocultured with BMSCs (p&lt0.05, fold change &gt1.5). When we analyzed the expression of these 13 genes in CLL cells from a cohort of 162 untreated patients by RNA-seq, we found associations between 4 genes (PNP, C16orf54, MOB3A, CDK2AP2) and clinical outcomes including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [multivariable Cox proportional hazards models, p&lt0.05]. Of the 4 genes, purine nucleoside phosphorylase (PNP), an enzyme in the purine pathway, had the most significant association with OS, PFS, and TTFT. Metabolomic profiling indicated an increased level of purine pathway metabolites adenosine, adenine, inosine, and hypoxanthine in cocultured CLL cells. WB analysis showed variable baseline PNP protein expression with 2 cohorts: low /no (L) and high (H) PNP using CLL blood cells. An induction in PNP protein levels were noticed in CLL cells expressing only LPNP when cocultured. Given the association of HPNP with adverse clinical outcomes, we tested for L vs. HPNP cohorts drug sensitivity to known BTK inhibitors(is) or Bcl2is. Treatment of CLL cells from untreated patients with Bcl2is venetoclax, S55746, and LP-118 (Bcl2-Bclxli, Newave) showed increased sensitivity to the Bcl2/Bclxlis in HPNP but not in LPNP cells. In addition, BTKis showed no difference in drug sensitivity associated with CLL PNP levels. To explain this discrepancy, WB and IP analyses were conducted in L vs. HPNP leukemic cells for apoptotic profiles, where we found increased phosphorylated-Bcl2(S70) and an increased association of Bcl2 with Bax in LPNP-expressing CLL cells compared to HPNP. These findings support increased resistance to apoptosis afforded by inhibiting Bcl2 for LPNP vs. HPNP-expressing CLL cells. Our future goals are to continue to understand the prognostic significance of the 4 genes with adverse clinical outcomes and to investigate how best to exploit the peculiar, enhanced sensitivity to Bcl2/Bclxlis found in the HPNP CLL cells. Citation Format: Sutapa Sinha, Weiguo Han, Zhiquan Wang, Brianna M. Finke, Heather C. Darby, Kari G. Rabe, Susan L. Slager, Chantal E. McCabe, Daniel R. O'Brien, Sameer A. Parikh, Esteban Braggio, Yi Chen, Fenlai Tan, Stephen P. Anthony, Yu Chen, Bing Dai, Yue Shen, Neil E. Kay. CLL drug resistance- A multifaceted mechanism [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 4423.

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.