Kay, Neil E.

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.

Purpose Venetoclax-based treatment for chronic lymphocytic leukemia (CLL) can be a logistically burdensome regimen due to extensive required monitoring of tumor lysis syndrome (TLS). The purpose of this study was to evaluate the feasibility and safety of pharmacist and registered nurse (RN) driven management of venetoclax ramp-up in CLL. Methods This was a prospective, pilot program for patients receiving venetoclax-based therapy for CLL. Patients had nine weekly visits during the first three cycles of therapy, of which five visits were independently performed by a pharmacist or RN. Laboratory tests to evaluate TLS were ordered and monitored independently by the RN in collaboration with clinicians (physician, nurse practitioner, physician assistant). Results Thirty CLL patients, median age 67 years, 73% treated in the frontline setting, started venetoclax between May 2022 and January 2023. After the initial three weeks of obinutuzumab treatment, TLS risk prior to venetoclax initiation had improved to low (93%), medium (3%), and high (3%). There was zero incidence of laboratory or clinical TLS by Howard criteria during the venetoclax ramp-up for all patients. Pharmacist and RNs performed 139 independent visits and monitored 197 sets of TLS labs. Conclusion Use of independent pharmacist and RN visits for toxicity checks and TLS monitoring during weekly venetoclax dose ramp-up did not lead to increased incidence of TLS in patients with CLL. These visits increased both access and efficiency within the CLL clinic. This concept has the potential to be applied in other cancer care settings with a variety of treatment regimens.

BACKGROUND Our knowledge of how the genomic landscape of chronic lymphocytic leukemia (CLL) is impacted by the selective pressure imposed by chemotherapy and targeted agents remains incomplete. We hypothesize that the extent of clonal evolution, baseline transcriptional state, and the overall and clone-specific growth kinetics of CLL will vary under native conditions or following exposure to chemotherapy and targeted therapy. METHODS We report the systematic genomic analysis of leukemia-normal whole-exome sequencing (WES) of 420 samples collected from 170 patients who were >65 years of age with treatment-naïve CLL. The treatment-cohort consisted of 83 patients who received ibrutinib (420mg daily) and 40 patients who received chlorambucil (0.5 mg/kg up to maximum 0.8 mg/kg, on days 1 and 15 of a 28-day cycle, up to 12 cycles) as part of the RESONATE-2 trial (NCT01722487). The ‘watch and wait’ (WW) cohort comprised 47 age-matched patients whose samples originated from the CLL Research Consortium and were untreated for their CLL during sampling. Sampling occurred at baseline and at day 300, and/or day 600, following initiation of therapy or following a diagnosis of CLL for those in the WW cohort. The WW cohort had more cases with a mutated IGHV (72% vs 40%), del17p (14% vs 0%), and del13q (72% vs 45%) when compared to the treatment cohort. Baseline CLL RNA-seq data were available for 150 patients. We used PhylogicNDT to reconstruct the clonal architecture from WES. By determining the fraction of cancer cells with each mutation (cancer cell fraction [CCF]) and their multiplicities (number of DNA molecules carrying a mutation per cancer cell), subclones were identified through clustering of CCFs of mutational events across samples from each patient. This allowed us to estimate the phylogenetic relationships and significant clonal shifts (defined as a distribution shift of CCF in a subclone of >95% between two timepoints, corresponding to a median ΔCCF of 18%) for each patient's CLL and to relate these shifts to somatic and clinical features. RESULTS In total, we identified 791 subclonal clusters across the three patient cohorts (ibrutinib = 406, chlorambucil = 189, WW = 196). We observed higher rates of significant clonal shifts between any two time points in patients exposed to chlorambucil (68%, q=0.008) and ibrutinib (77%, q<0.0001) when compared to patients on WW (36%). There was no significant difference in the number of patients exhibiting a clonal shift between those receiving ibrutinib and chlorambucil (q=0.3). More patients treated with ibrutinib (54%, q=0.0002) and chlorambucil (50%, q=0.004) harbored a clone that reduced in size than those on WW (17%). More patients treated with ibrutinib (71%, q<0.0002) and chlorambucil (55%, q=0.12) possessed at least one clone that increased in size during therapy than patients receiving WW (34%). In ibrutinib-treated patients, SF3B1 mutations (q=0.13, 14% vs 5%) and XPO1 mutations (q=0.13, 5% vs 0.3%) were enriched in subclones that significantly reduced over the complete time period. We also observed more BIRC3 mutations in subclones that had a significant shift in CCF when compared to subclones that remained stable over time (q=0.2, 5% vs 2%). Consistent with the overall study, with 8 years of follow-up in this patient subset, long-term progression-free survival (PFS) was significantly higher in the patients treated with ibrutinib when compared to chlorambucil (P<0.0001). This PFS benefit was also seen regardless of subclonal growth patterns. Using baseline RNA-seq data, we identified 7 of the 8 recently defined CLL expression clusters (EC), that were identified in the context of chemoimmunotherapy for CLL (Knisbacher et al. Nat Gen 2022), and could recapitulate the relationship to outcomes in individuals treated with chlorambucil. However, despite the larger cohort, no significant relationship between EC clusters and PFS were observed in ibrutinib-treated patients, suggesting that a distinct predictive baseline transcriptional signature may be present in ibrutinib-treated patients. CONCLUSION Our analyses highlight the need to interrogate the molecular features of CLL in relation to the impact of therapeutic selective pressure imposed by BTK inhibition, which appear distinct from exposure to chemotherapy. Ongoing analyses aim to identify relationships between patterns of growth and subclonal growth rates molecular and clinical features.

Background: Monoclonal B-cell lymphocytosis (MBL) is a common premalignant condition that can be classified into chronic lymphocytic leukemia (CLL)-type (CD5, CD23, and CD20 co-expression), and non-CLL-type (CD5-negative) based on flow cytometry immunophenotypes. CLL-type MBL is relatively well characterized with increased risk of hematologic (especially lymphoid) malignancies, serious infections, and decreased response to vaccinations. However, the prevalence, genetic predispositions, and clinical consequences of non-CLL-type MBL remain to be fully elucidated. Herein, we report findings in non-CLL-type MBL in a large screening cohort. Methods: We screened 10,330 individuals (40 years or older with no prior history of hematologic cancers) for MBL with highly sensitive flow cytometry, in the Mayo Clinic Biobank, with patients recruited from primary care-based clinics. Individuals were genotyped by Regeneron Genetics. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Among those individuals residing in the 27 counties surrounding Rochester, Minnesota, we identified incident hematologic malignancies and melanomas using ICD codes and the Rochester Epidemiology Project, a population based medical records linkage system. All diagnoses were manually reviewed for confirmation. Cox regression was used to calculate hazard ratios (HR) and 95% CIs, adjusted for age and sex. Results: We identified 1,736 individuals with MBL and 8,341 individuals without MBL. Among those with MBL, 246 (14.2%) had non-CLL-type MBL as the major clone. The median age of non-CLL-type MBL was 77 years (range, 47-95). The prevalence of non-CLL-type MBL significantly increased with age, with 0.5% prevalence among those between 40 to 59 years of age, 2.3% prevalence among those between 60 and 79 years, and 7.8% prevalence in those 80 years of age or older. The prevalence of non-CLL-type MBL was significantly higher (P < .001) among males (3.1%) than females (2.0%). Using the genome-wide complex trait analysis tool, the heritability of non-CLL-type MBL was estimated to be ~26%, suggesting inherited genetics plays a role in etiology. To identify common inherited variants, we first evaluated the known CLL susceptibility variants, many of which also associated with CLL-type MBL. Among the 42 variants, none were statistically associated (all P > .05) with non-CLL-type MBL. We next conducted a genome-wide association study to identify novel variants, and the most significant variant resided in 10q26.3 (minor allele frequency [MAF]control = 0.7%, MAFMBL = 3.5%, OR = 6.4, 95% CI 3.6-11.3, P = 5.7 * 10-8). Among the 6,478 individuals (192 with non-CLL-type MBL and 6,286 without MBL) followed for incident hematologic malignancies, 36 developed lymphoid and 23 developed myeloid malignancies after a median of 4.2 years of follow up. Similar to CLL-type MBL, non-CLL-type MBL was associated with a 5.2-fold risk for lymphoid malignancies (95% CI 2.0-13.0, P < .001). Elevated but not significant risks were observed for myeloid malignancies (HR = 2.3, 95% CI 0.5-10.3, P = 0.27). The lymphoid malignancies developed by individuals with non-CLL-type MBL included diffuse large B-cell lymphoma, multiple myeloma, marginal zone lymphoma, and other low-grade lymphomas. We recently reported that individuals with CLL-type MBL are at higher risk for developing melanoma (HR = 1.9, 95% CI 1.3-2.8). Interestingly, non-CLL-type MBL had no evidence of an association with melanoma risk (HR = 0.4, 95% CI 0.1-1.8). We did not observe a significant difference in overall survival between individuals with non-CLL-type MBL vs those without MBL. Conclusions: In the largest screening cohort to date, we evaluated the etiology and clinical outcomes of non-CLL-type MBL, an understudied yet common condition that occurs in ~4 million adults in the United States. Non-CLL-type MBL is more common in men and older individuals, and evidence supports that inherited genetics plays an etiological role with 10q26.3 locus identified as a novel susceptibility locus. Individuals with non-CLL-type MBL had significantly increased risk of lymphoid malignancies, similar to CLL-type MBL, but, distinct from CLL-type MBL, no evidence of an increased risk of melanoma. Our study provides insights into the prevalence, genetic predispositions and clinical consequences of non-CLL-type MBL and how it differs from the classic CLL-type MBL.

Introduction: Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), changed the treatment landscape by demonstrating improved overall survival (OS) compared with chemotherapy/chemoimmunotherapy across multiple phase 3 trials in patients (pts) with CLL, including those with high-risk genomic features. Recently, data were reported for pts treated with ibrutinib in the phase 3 RESONATE-2 trial with up to 10 years of follow-up, representing the longest follow-up of any BTKi used in first-line treatment of CLL. With the most robust long-term follow-up data among BTKis, ibrutinib therapy trials are uniquely positioned to assess the long-term OS benefit of first-line BTKi treatment in pts with CLL across pt subgroups. We previously demonstrated that first-line ibrutinib is associated with OS rates that are similar to those in the age-matched general population, with a median follow-up of 5.9 years since initial diagnosis (Ghia P et al. HemaSphere 2024), but it remains unclear whether this holds true for pts with high-risk genomic features. This updated analysis evaluated long-term OS outcomes with a median follow-up of 8 years since randomization and 10 years since initial diagnosis in a pooled population of pts with previously untreated CLL, including those with high-risk features, who received first-line ibrutinib treatment in 2 phase 3 trials with comparison of survival estimates to the US age-matched general population. Methods: Data were pooled for pts who received first-line treatment with single-agent ibrutinib or ibrutinib + rituximab in the RESONATE-2 (NCT01722487) and ECOG-ACRIN E1912 (NCT02048813) trials, respectively. OS probabilities from the time of randomization and from the time of initial diagnosis for ibrutinib-treated pts were compared with an age-matched general population using 2021 life tables for the total US population published by the Centers for Disease Control and Prevention using the Kaplan-Meier method. Subgroup analyses were performed for high-risk pts, defined as those with del(11q), del(17p), mutated TP53, and/or unmutated IGHV (uIGHV). Results: A total of490 pts were pooled across the 2 studies: 352 pts (71.8%) were treated with ibrutinib + rituximab, and 135 pts (27.6%) were treated with single-agent ibrutinib; 3 pts did not receive study treatment. The median age at time of randomization was 61 years; 36.5% (179/490) were aged ≥65 years, 21.8% had del(11q), 7.8% had del(17p) and/or mutated TP53, and 54.7% had uIGHV. The median time from initial CLL diagnosis to randomization was 20.9 months (range, 0.0-341.8); the median follow-up was 123.5 months (10.3 years) from initial diagnosis and 99.2 months (8.3 years) from the time of randomization. From the time of randomization, estimated 9-year OS rates were 81.2% (95% CI, 76.8-84.9) in all ibrutinib-treated pts versus 82.0% (95% CI, 78.3-85.2) in the age-matched population (hazard ratio [HR] 1.17; 95% CI, 0.86-1.58). In high-risk pts, estimated 9-year OS rates were 79.5% (95% CI, 73.5-84.3) for ibrutinib-treated pts versus 83.2% (95% CI, 78.5-86.9) in the age-matched general population (HR 1.24; 95% CI, 0.84-1.84). In pts aged ≥65 years, estimated 9-year OS rates were 68.7% (95% CI, 60.5-75.6) for ibrutinib-treated pts and 68.7% (95% CI, 61.4-75.0) in the age-matched general population (HR 1.12; 95% CI, 0.77-1.63). In pts aged <65 years, estimated 9-year OS rates were 89.3% (95% CI, 84.6-92.7) for ibrutinib-treated pts and 90.0% (95% CI, 86.1-92.9) in the age-matched general population (HR 1.15; 95% CI, 0.68-1.94). From the time of initial diagnosis, estimated 15-year OS rates were 78.4% (95% CI, 72.8-83.0) in all ibrutinib-treated pts versus 72.0% (95% CI, 67.8-75.7) in the age-matched general population (HR 0.83; 95% CI, 0.62-1.11). Study treatment had been discontinued in 59.8% (293/490) of pooled ibrutinib-treated pts; the most frequent reasons for discontinuation were adverse events (43.0%; 126/293) and progressive disease (20.5%; 60/293). Study treatment was ongoing in 31.8% of pooled pts (156/490). Conclusions: With the longest follow-up time for any commercially available targeted therapy, this pooled analysis demonstrates that, regardless of evaluation from randomization or initial diagnosis, and irrespective of age or high-risk features, first-line treatment with ibrutinib provides long-term OS benefit with survival estimates that appear similar to those of a US age-matched cohort.

Background: Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL), and several studies suggest that hypogammaglobulinemia at the time of CLL diagnosis may predict shorter time to first therapy (TTFT; Parikh et al, 2015). However, it is unclear if hypogammaglobulinemia can predict TTFT independent of the CLL-International Prognostic Index (CLL-IPI) and the tumor mutational load (TML). We sought to determine if serum immunoglobulin (Ig) levels at diagnosis can independently predict outcomes in patients with CLL and its precursor, high-count monoclonal B-cell lymphocytosis (MBL). Methods: We used the Mayo Clinic CLL Database to identify newly diagnosed CLL/MBL consented within 2 years of diagnosis. Next generation sequencing (NGS) was performed using a 59 gene panel (SureSelect) to detect mutated CLL genes. Serum Ig levels were quantitated by radial immunodiffusion (Immunoplates). Serum Ig levels below the lower limit of normal were defined as hypogammaglobulinemia. We used Chi-squared or Fisher's exact tests to compare discrete variables and the Kruskal Wallis test for continuous variables. TTFT was calculated from date of diagnosis to date of first treatment or date last known to be untreated. We used Cox regression models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for TTFT and overall survival (OS) associations. Results: We identified 895 newly diagnosed patients (588 CLL and 307 MBL) who had undergone NGS for recurrent somatic mutations and had serum Ig levels available at the time of diagnosis. The median absolute B-cell count was 6.7 x109/L; 613 (69%) were male, 375 (42%) had unmutated IGHV genes, and 81 (9%) had TP53 disruption. Based on CLL-IPI, 412 (46%) patients had low risk, 285 (32%) had intermediate risk, and 198 (22%) had high/very high risk. The most commonly mutated genes were NOTCH1 (14%), SF3B1 (11%), TP53 (10%), NFKBIE (8%), ATM (6%), and BIRC3 (6%). Based on the number of genes with high impact or hotspot mutations, we classified 40%, 29%, and 31% patients with TML scores of 0, 1, or 2+, respectively. At CLL/MBL diagnosis, the median serum IgG, IgA, and IgM was 891 mg/dL (111-2620), 121 mg/dL (1-1150), and 44 mg/dL (5-1320), respectively; 284 (32%) patients had low serum IgG, 128 (17%) had low serum IgA, and 322 (42%) had low serum IgM. Low serum IgA was associated with mutations in NOTCH1, SF3B1, ATM, BIRC3 and NFKBIE, low IgM was associated with SF3B1 and BIRC3 mutations (all comparisons, P < .05), and low IgG was associated with BIRC3 mutation (P = .05). The median follow-up for the cohort was 8.4 years. A total of 359 patients progressed requiring therapy (299 CLL and 60 MBL), and 265 patients died (177 CLL and 88 MBL). The TTFT was significantly shorter for patients with low serum IgG compared to normal serum IgG at diagnosis (median 4.4 vs 10.8 years) and similar findings were observed in individuals with low serum IgA (1.7 vs 10.8 years) and those with low serum IgM (4.6 vs 11.1 years). On univariable analysis, the following were associated with a shorter TTFT: low serum IgG (HR: 1.8, 95% CI 1.4-2.2), low serum IgA (HR: 3.5, 95% CI 2.7-4.4), low serum IgM (HR: 1.9, 95% CI 1.5-2.3), TML score of 1 (HR: 1.7, 95% CI 1.3-2.2), TML of 2+ (HR: 4.4, 95% CI 3.4-5.7), CLL-IPI intermediate risk (HR: 3.7, 95% CI 2.8-4.8), and CLL-IPI high/very high risk (HR: 7.6, 95% CI 5.8-10.1) (all comparisons, P < .0001). In multivariable analyses after adjusting for CLL-IPI and TML, low serum levels of IgG (HR: 1.8, 95% CI: 1.5-2.3), IgA (HR: 2.4, 95% CI: 1.8-3.0), and IgM (HR: 1.8, 95% CI: 1.5-2.3) were independently associated with a shorter TTFT (all comparisons, P < .0001). No significant differences in OS were detected between patients with low and normal serum IgG, IgM, and IgA on univariate analysis. Similar results for TTFT and OS were observed after adding clonal B-cell count to the Cox model. Conclusions: Our study shows that low serum Ig levels at the time of initial diagnosis is predictive of TTFT independently of the CLL-IPI and TML, which can further risk stratify individuals with newly diagnosed CLL/MBL. Additionally, low serum IgA, IgM, and IgG levels at diagnosis are significantly associated with specific somatic mutations in individuals with newly diagnosed CLL/MBL.

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.

Introduction Venetoclax + rituximab resulted in a 2-year progression-free survival estimate of 85% in the mostly 2nd-line, post-chemotherapy setting of the MURANO clinical trial (Seymour NEJM 2018). Short remissions with venetoclax in patients (pts) with prior cBTKi treatment (tx) for CLL are reported. However, these cohorts have included heavily pre-treated pts. Here, we report outcomes with venetoclax-based regimens in a contemporary cohort with cBTKi-exposed CLL and no prior chemotherapy. Methods We conducted a retrospective study of pts who received venetoclax for relapsed CLL after prior cBTKi tx at Mayo Clinic and across 8 centers in France. Pts with any prior chemotherapy tx were excluded. Pts were grouped as cBTKi-resistant (CLL disease progression on cBTKi) and cBTKi-exposed (any prior cBTKi tx but without disease progression). Overall survival (OS) was analyzed as the time from initiation of venetoclax until date of death or last known to be alive. Time to next therapy or death (TTNTD) after venetoclax was defined as the time from venetoclax initiation until the earliest date of next tx or death. Cox proportional hazards regression models were used to estimate associations of factors with OS and TTNTD. Results A total of 85 pts received venetoclax, including: venetoclax monotherapy (n=13, 15%), venetoclax + rituximab (n=45, 53%), and venetoclax + obinutuzumab (n=27, 32%). Patient characteristics at the time of venetoclax start were as follows: median age 69 years (interquartile range [IQR] 62-77), 59 (69%) male, 45% (33/73 evaluated) with TP53 disruption (del(17p) or TP53 mutation), 82% (46/56 evaluated) with unmutated IGHV, and 41% (15/37 evaluated) with complex karyotype (CK; ≥3 clonal structural or numerical abnormalities on karyotype). The median prior lines of therapy was 1 (IQR 1, 2); 32 (38%) pts had received prior anti-CD20 monoclonal antibody and 8 (9%) pts had received 2 prior cBTKi. Prior cBTKi tx was stopped for a reason other than disease progression in 46 (54%) pts (cBTKi-exposed) and due to CLL progression in 39 (46%) pts (cBTKi-resistant). TP53 disruption, unmutated IGHV, and CK were more common in the cBTKi-resistant subgroup. Among pts with cBTKi-resistant CLL, 60% (12/20 evaluated) had a BTK and/or PLCG2 mutation. Median follow-up from start of venetoclax-based therapy was 20 months. The median TTNTD and OS for the entire cohort was 36 (95% CI 29-not evaluable [NE]) and 68 (95% CI 39-NE) months, respectively. Among the 46 pts with cBTKi-exposed CLL, the median duration of prior cBTKi tx was 6.7 months (IQR 1.8, 23.7). The median TTNTD and OS from venetoclax start were both 78 months in pts with cBTKi-exposed CLL. The 24-month TTNTD and OS estimates were 73% and 81%, respectively. Among the 39 pts with cBTKi-resistant CLL, the median duration of prior cBTKi tx was 42.3 months (IQR 23.4, 60.1). The median TTNTD from venetoclax start in pts with cBTKi-resistant CLL was 30 months. The 24-month TTNTD estimate was 65% in the cBTKi-resistant subgroup, 53% with venetoclax monotherapy (n=5), 62% with venetoclax + rituximab (n=30), and 100% venetoclax + obinutuzumab (n=4). The median OS from venetoclax initiation was 39 months overall in pts with cBTKi-resistant CLL. The 24-month OS estimate was 84% in the cBTKi-resistant subgroup overall, 53% with venetoclax monotherapy, 87% with venetoclax + rituximab, and 100% with venetoclax + obinutuzumab. On a univariate analysis in the whole cohort, only TP53 disruption was associated with shorter TTNTD (HR 2.23; 95% CI 1.02-4.87; P=0.04) and only older age was associated with shorter OS (HR 1.05; 95% CI 1.007-1.095, P=0.02). On a univariate analysis in the cBTKi-resistant subgroup, only TP53 disruption was associated with shorter TTNTD (HR 6.68; 95% CI 1.51-29.46; P=0.01) and OS (HR 7.95; 95% CI 1.02-61.97; P=0.04). Duration of prior cBTKi exposure (as a continuous variable) was not associated with survival outcomes in the cBTKi-resistant subgroup. Conclusions Pts with chemotherapy-naïve, cBTKi-treated CLL had worse than expected outcomes with venetoclax-based tx in a mostly 2nd-line tx cohort, particularly pts with cBTKi-resistant CLL. The frequency of high-risk disease features, shorter duration of disease control with their initial cBTKi tx, and small cohort size warrant caution in generalizing these results to all pts with cBTKi-resistant disease. Novel tx trial strategies in 1st- and 2nd-line settings remain a high priority for high-risk pts.