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Introduction: Cyclin D1, encoded by the CCND1 gene, is primarily known as a major regulator of cell cycle progression. Emerging studies have demonstrated a role for cyclin D1 in regulating gene transcription. In mantle cell lymphoma with t(11;14), cyclin D1 strongly binds to the promoters of highly transcribed genes and its overexpression led to genome-wide transcriptional downregulation. However, the transcriptional role for cyclin D1 in t(11;14) MM remains undefined. This study aims to elucidate the global chromatin and transcriptional changes induced by cyclin D1 in t(11;14) MM. Methods: Knockout (KO) of CCND1 in U266B1, a MM cell line carrying t(11;14), was performed by the Synthego (Redwood City, CA) using CRISPR/Cas9, which generated a frameshift insertion of base pair (bp) “A” after 69,641,478 bp within exon 1. Two independent KO clones were used for downstream analysis. ChIP-seq and ATAC-seq were performed for KO (n=2) and wild-type clones (WT, n=2). ChIP-seq used antibodies against H3K4me3 (promoter) and H3K4me1 and H3K27ac (enhancer). Libraries were sequenced to 2x51 bp on the Illumina NextSeq 2000 platform. Total RNA libraries (in triplicate) were sequenced to 2x150 bp. ChIP-seq and ATAC-seq reads were mapped to hg38 with BWA, and peaks were identified by MACS2 at FDR 0.01. RNA-seq reads were mapped to hg38 with STAR and gene raw counts were estimated using featureCounts. Differential peaks and differentially expressed genes between KO and WT were identified using edgeR at FDR 0.05 and fold-change 2. Only peaks present in at least 2 samples and genes having reads-per-million value 0.1 in at least 1 sample were included, excluding those from chrY. Pathway analysis was performed with the Enrichr package. Results: Differential expression analysis identified 2-fold more genes that were down-regulated than up-regulated in KO compared to WT (467 vs. 233). Pathway analysis revealed an enrichment of TNF-alpha signaling via NF-kappaB, inflammatory response, and apoptosis in association with down-regulated genes, while up-regulated genes are enriched in myogenesis, KRAS signaling, and epithelial mesenchymal transition pathway. In parallel, differential chromatin analysis identified 2.5% (1,161) of the peaks from H3K27ac and 0.5-1% (359-610) of the peaks for the other 3 marks that showed significant changes of signal levels between KO and WT. For the promoter mark H3K4me3, there are largely a comparable number of peaks showing increased (191 peaks) and decreased (168 peaks) signals, respectively, in KO compared to WT. However, for ATAC-seq and H3K4me1, about 70% of the differential peaks showed an increased signal in KO, in contrast with H3K27ac for which 60% of differential peaks showed decreased signal in KO. The preferential loss of H3K27ac occupancy is consistent with the observation of a higher proportion of genes being down-regulated in KO. These results indicated that knockout of CCND1 led to a global increase of chromatin accessibility, but a reduction of H3K27ac and gene expression. Further annotation revealed that, for H3K27ac and H3K4me1/ATAC-seq, 92% and 97% of the differential peaks were located >2kb away from the transcription start sites (TSSs) of expressed genes. Even for H3K4me3, 88% of the differential peaks are >2kb away from TSSs. The results suggest that the changes of chromatin upon CCND1 knockout predominantly occurred at distal regulatory regions. For up- and down-regulated genes, we estimated ChIP-seq and ATAC-seq signals in the TSS 2kb regions, revealing that up-regulated genes were overall associated with increased signals. Further analysis found that, for ATAC-seq and H3K4me3, 44% and 59% of the differential peaks located in the promoter regions were associated with differentially expressed genes, which dropped to ~20% for the two enhancer marks. These data indicate that changes in chromatin state impact the expression of the associated genes. Conclusion: Knockout of CCND1 impacts local chromatin state, particularly at enhancer regions, and the transcriptional program. In promoter-proximal regulatory regions, the changes of chromatin accessibility and H3K4me3 are strongly correlated with the expression changes of nearby genes. Further studies will identify the genes targeted by the differential enhancers and their possible roles in MM pathogenesis mediated by cyclin D1 following the t(11;14) event.

Introduction MYD88 L265P mutations (MUT) are found in 90% of patients with Waldenstrom macroglobulinemia (WM) and lead to the activation of pro-survival signaling. Limited literature has demonstrated that patients with MYD88 wild-type (WT) WM have poorer responses to conventional therapies and a higher likelihood of histological transformation. Increasingly the role of non-coding genomics in lymphomagenesis has demonstrated importance and currently, the epigenetic landscape of MYD88 WT WM remains underexplored. This study utilized a multi-omics approach to assess DNA methylation in MYD88 WT WM compared to MYD88 MUT patients and controls. Methods Bone marrow samples were prospectively collected from 20 subjects [3 MYD88 WT/CXCR4 WT WM, 12 MYD88 MUT WM and 5 normal controls] and positive selection for CD19+ and/or CD138+ B cells was performed. The analysis focused on the comparison between MYD88 WT WM to normal controls and MYD88-WT to MUT WM. Genome-wide DNA methylation analysis was performed, and CpG methylation ratios were segmented into 200-bp regions. Differentially methylated regions (DMRs) were determined using a Q-value <0.05 and absolute methylation difference ≥10%. Next, RNA-seq data was interrogated and differentially expressed genes (log2fold change>0.5 or <-0.5, false discovery rate<0.05) with corresponding promoter hypo- or hypermethylation were assessed. Ingenuity Pathway Analysis was used to determine potentially epigenetically regulated pathways using a corrected p<0.05. Results Comparing MYD88 WT WM samples to controls, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (29129 vs 6300), 3'untranslated region (UTR) (2068 vs 1551), 5'UTR (10169 vs 735), intron (30344 vs 24511), exon (2224 vs 6855), CpG island (30372 vs 1240) and CpG shore (14477 vs 5872) (all p<0.001). Next, mRNA analysis demonstrated that 214 genes were differentially up-/downregulated with concordant promoter hypo-/hypermethylation. On the assessment of promoter-methylation-based pathways, multiple pathways involved in cell signaling, proliferation, gene regulation, cytokine and immune signaling and metabolism were found to be deregulated. Most pathways were found to be negatively enriched in MYD88 WT patients compared to controls including several members of interleukin (IL) signaling (IL-2, IL-3, IL,7) and regulators of cell proliferation and signaling including mTOR, PI3K/AKT, and p53. Positive enrichment of the PTEN, FGFR3 and ERK/MAPK pathways were observed. Underlying multiple pathways was promoter hypermethylation with corresponding mRNA downregulation of AKT3 and PIK3CA. Furthermore, promoter hypomethylation with transcript upregulation of PTEN was observed. Next, in comparison of MYD88 WT to MUT samples, a statistically greater number of hypermethylated DMRs were observed across genomic regions (hyper- vs hypomethylated): promoter (24829 vs 2544), 3'UTR (3534 vs 378), 5'UTR (6111 vs 605), intron (52292 vs 5360), exon (21066 vs 2187), CpG island (18334 vs 1551) and CpG shore (16351 vs 1957) (all p<0.001). Assessing mRNA expression, 354 genes were up-/downregulated with concordant promoter hypo-/hypermethylation. Pathway analysis revealed positive enrichment of the mitotic G1 phase to G1/S transition pathway with upregulation of the CDK4, LYN, and PSMA6 transcripts in MYD88 WT compared to MUT samples. Further, negative enrichment of cell signaling regulators including the RHO GTPase family, IL pathways (IL-4, IL-8, IL-13, IL-17) and integrin signaling were found in MYD88 WT compared to MUT samples. Underlying multiple pathways were downregulation with promoter hypermethylation of BCL2, an important regulator of apoptosis. Conclusion Genome-wide methylation analysis of MYD88 WT WM revealed a unique DNA methylation profile, with potential epigenetic alteration of key oncogenic transcripts and pathways. The negative enrichment of cell signaling and cytokine/inflammatory pathways in MYD88 WT samples compared to controls and MYD88 MUT WM suggests an epigenetic mechanism for immune evasion and potentially underlies the reduced response to conventional WM-directed therapies. Overall, this study reveals that MYD88 WT WM is epigenetically distinct from MYD88 MUT WM and regulators such as methylation may underlie the unique clinical and phenotypic properties observed.

PURPOSE Chronic lymphocytic leukemia (CLL)–phenotype monoclonal B-cell lymphocytosis (MBL) is a premalignant condition that is roughly 500-fold more common than CLL. It is unknown whether the two-fold increased risk of developing melanoma associated with CLL extends to individuals with MBL. METHODS Using the Mayo Clinic Biobank, we identified participants who were 40 years or older with no previous hematological malignancies, who resided in the 27 counties around Mayo Clinic, and who had available biospecimens for screening. Eight-color flow cytometry was used to screen for MBL. Individuals with MBL were classified as low-count MBL (LC-MBL) or high-count MBL on the basis of clonal B-cell percent. Incident melanomas were identified using International Classification of Diseases codes and confirmed via medical records review. Cox regression models were used to estimate hazard ratios (HRs) and 95% CI. RESULTS Of the 7,334 participants screened, 1,151 were identified with a CD5-positive MBL, of whom 1,098 had LC-MBL. After a median follow-up of 3.2 years (range, 0-13.5), 131 participants developed melanoma, of whom 36 individuals were positive for MBL. The estimated 5-year cumulative incidence of melanoma was 3.4% and 2.0% among those with and without MBL, respectively. After adjusting for age, sex, and history of previous melanoma, individuals with MBL exhibited a 1.86-fold (95% CI, 1.25 to 2.78) risk of melanoma. This elevated risk persisted when analysis was restricted to those without a history of melanoma (HR, 2.05 [95% CI, 1.30 to 3.23]). Individuals with LC-MBL had a 1.92-fold (95% CI, 1.29 to 2.87) increased risk of developing melanoma overall and a 2.74-fold increased risk (95% CI, 1.50 to 5.03) of melanoma in situ compared with those without MBL. CONCLUSION LC-MBL is associated with an approximately two-fold increased risk of melanoma overall and a 2.74-fold increased risk of melanoma in situ.

Abstract Currently, the role of DNA methylation in the immunoglobulin M (IgM) monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenström macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

7011 Background: Monoclonal B-cell lymphocytosis (MBL) is a precursor to chronic lymphocytic leukemia (CLL) but also a risk factor for other lymphoid malignancies. Clonal hematopoiesis (CH), originally defined as the presence of mutations in myeloid driver genes, is a precursor to myeloid malignancies. Recently, CH that includes genes associated with lymphoid malignancies has been reportedly associated with risk of lymphoid malignancies, and particularly CLL. Little is known about the association between CH and MBL and their joint effects on risk of hematological malignancies (HM). Methods: Study participants were from the Mayo Clinic Biobank, a large-scale biorepository of patients who provided a peripheral blood sample. CH at enrollment was determined using whole-exome sequencing (50x coverage). Myeloid-CH (M-CH) was based on mutations located in 56 genes associated with myeloid malignancies. Lymphoid-CH (L-CH) was based on 235 genes associated with lymphoid malignancies. Individuals were screened for MBL using eight-color flow cytometry in two different cohorts of Biobank participants. Cohort 1 (N=3883) included participants who had available sample for MBL screening at enrollment. Cohort 2 (N=5684) were participants recontacted to provide a sample for MBL screening. Incident HM were identified using ICD codes and confirmed via medical record review. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Cox regression was used to estimate hazard ratios (HR), with time defined as date between MBL sample and the first of incident HM, death, or 9/30/2023. Analyses were adjusted for age at MBL screening and sex. Results: In total, 9567 individuals (39% male, median age 66 years) were screened for MBL with available CH, of whom 17% were positive for MBL, 7% were positive for M-CH, and 2% were positive for L-CH. We found no evidence of an association between M-CH and MBL (OR=1.11, 95% CI:0.91-1.34) nor L-CH and MBL (OR=1.05, 95% CI:0.70-1.55). When subset to Cohort 1, where the same sample was used for both CH and MBL screening, the results held (M-CH OR=1.11, 95% CI: 0.83-1.48; L-CH OR=0.82, 95% CI:0.40-1.53). Next, we investigated the effect of each of these precursors with incident HM. Median follow-up was 4 years, and 78 individuals developed incident HM (31 myeloid, 49 lymphoid overall, 8 CLL). When modeling both precursors, both L-CH (HR=6.23, 95% CI:2.46-15.79) and MBL (HR=3.89, 95% CI:2.17-6.99) were independently associated with incident lymphoid malignancy. When excluding CLL events, the association held (L-CH: HR=5.94, 95% CI:2.11-16.77; MBL: HR=2.95, 95% CI:1.54-5.66). Only M-CH was significantly associated with incident myeloid malignancies. Conclusions: In the largest cohort with MBL and CH precursors measured, we found no evidence of an association between them. However, both MBL and L-CH were strong independent risk factors for incident lymphoid malignancies.

Abstract Introduction: Deletion in the short arm of chromosome 17 (del17p) is a strong, negative prognostic biomarker found in about eight percent of patients with untreated chronic lymphocytic leukemia (CLL). CLL patients with del17p exhibit resistance to therapy, accelerated disease progression, and global immunodeficiency. It is unclear the extent to which global immunodeficiency impacts disease course and therapy outcomes. The goal of this study was to document the blood immune repertoire of patients with untreated CLL who have del17p and associate with clinical features, time to first treatment (TFT), and overall survival (OS). Methods: Biobanked blood samples from treatment-naïve del17p CLL patients (n = 58) and age-matched controls (n = 5) were obtained. B cells and T cells were analyzed with comprehensive, cell type-specific flow cytometry panels and visualized with unbiased dimensionality reduction. Lab values were log-transformed and compared using t-tests and one-way ANOVAs. TFT was calculated from date of sample collection to date of first CLL-directed therapy or last known untreated date; OS was calculated from date of sample collection to death or last known alive status. Cox proportional hazards regression analyses (results reported as hazard ratio [HR] and 95% confidence interval [CI]) were employed to identify associations of lab values with OS and TFT. Results: Patients with del17p CLL had increased proportions of T regulatory cells (Tregs) among total CD4+ T cells compared to controls (CLL 11.1%, control 5.7%, p = 0.05). A higher proportion of CD8+ T cells was associated with longer TFT (HR 0.57, 95%CI 0.36-0.89); in contrast, higher proportions of TIGIT+ (HR 1.83, 95%CI 1.03-3.28) and TIM3+ conventional (non-Treg) CD4+ T cells were associated with shorter TFT (HR 1.70, 95%CI 1.02-2.84). A higher proportion of PD1+ CD8+ T cells was associated with shorter OS (HR 1.79, 95%CI 1.05-3.06), in addition to PD1+ conventional CD4+ T cell proportion (HR 2.21, 95%CI 1.06-4.60). CLL B cells expressed PD-1 and inducible T cell costimulator (ICOS) at significantly lower median fluorescence intensities (MFIs) than control B cells (p < 0.0001 for both). The CXCR5+ MFI (HR 1.0002, 95%CI 1.0001-1.003) and CD23+ MFI (HR 1.88, 95%CI 1.01-3.50) of CLL B cells were associated with shorter TFT. Discussion: In this cohort of treatment-naïve del17p CLL patients, we determined that patients with higher proportions of exhausted CD4+ and CD8+ T cell phenotypes had significantly shorter OS. Cytotoxic T cell proportion positively associated with TFT, whereas proportions of CD4+ T cell exhaustion phenotypes negatively associated with TFT. Higher CXCR5+ and CD23+ MFIs of CLL B cells also associated with shorter TFT. The preliminary findings from our study suggest that the blood immune repertoire has strong associations with clinically relevant outcomes such as time to first treatment and overall survival in patients with untreated del17p CLL. Citation Format: Ethan J. Katcher, Kim A. Gwin, Sameer A. Parikh, Kari G. Rabe, Petra K. Lothert, Preeti Trisal, Esteban Braggio, Susan L. Slager, Nicholas Strong, Daniel L. Van Dyke, Zhiquan Wang, C. Chris Huang, Anita K. Gandhi, Kay L. Medina, Neil E. Kay. Correlating the blood immune repertoire and clinical features of del17p CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB345.

Abstract Background: The impact of a prior history of non-hematologic malignancy (NHM) on clinical outcomes among individuals with newly diagnosed chronic lymphocytic leukemia (CLL) or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, is not well known. Methods: In this prospective study, we identified individuals with CLL/MBL who were seen in the Division of Hematology at the Mayo Clinic, Rochester. Participants provided consent within nine months of diagnosis, had no prior history of hematological malignancy, and completed a self-reported risk-factor questionnaire, including a prior history of NHM. Time to first treatment (TTFT) was defined as the time between the date of diagnosis and the earliest date of the first CLL treatment, death, or the last known treatment-free follow-up date. Overall survival (OS) was defined as the time from diagnosis to death or the last follow-up. We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations between a prior history of a NHM with OS and TTFT. We adjusted for sex and the CLL International Prognostic Index (CLL-IPI), a score comprised of five key CLL prognostic factors, including age and Rai stage. Results: Among the 1,090 CLL/MBL individuals, 833 (76%) had CLL. The median age at diagnosis was 64 years (range 24-89), with 65% being men, and 230 (21%) reported a prior history of NHM. The most common tumor was non-melanoma skin cancer (NMSC, 8.9%, 96 individuals), followed by prostate, testicular, and penile tumors (7.3%, 51 individuals), and melanoma (1.9%, 20 individuals). CLL/MBL patients with a prior history of a NHM were more likely to be over 65 years old (29%) than under 65 years old (14%, p < 0.0001). After a median follow-up of 2.9 years (range 0-21), 470 CLL/MBL patients progressed, requiring CLL treatment. CLL/MBL individuals with a prior history of NHM had a longer TTFT compared to those without (HR=0.69; 95% CI: 0.53-0.90; p=0.006). Individuals with a prior history of NHM who had either intermediate or high-very high CLL-IPI score had significantly longer TTFT (HR= 0.55; 95% CI: 0.33-0.92; p=0.02, and HR=0.65; 95% CI: 0.43-0.97; p=0.03, respectively) compared to those without NHM. After a median follow-up of 8.6 years (range 0-21), 359 individuals died. CLL/MBL patients with a prior history of NHM also experienced reduced OS compared to those without (HR=1.46; 95% CI: 1.08-1.97, p=0.013), particularly among those with low (HR=1.70; 95% CI: 0.96-3.00, p=0.07) or intermediate-risk (HR=2.08; 95% CI: 1.24-3.51, p=0.006), according to CLL-IPI. After excluding NMSC, results remained constant. Conclusions: At the time of diagnosis, one out of five CLL/MBL individuals had a prior history of a NHM. These individuals had a longer time to first CLL directed treatment and reduced OS, even after accounting for other important CLL prognostic factors, which deserves additional studies. Citation Format: Bryan A. Vallejo, Cristine Allmer, Kari G. Rabe, Dennis P. Robinson, Yucai Wang, Paul J. Hampel, Esteban D. Braggio, Neil E. Kay, James R. Cerhan, Sameer A. Parikh, Susan L. Slager. The impact of prior history of non-hematologic malignancies on time to first treatment and overall survival among individuals with CLL or MBL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7450.

Abstract The treatment of Chronic lymphocytic leukemia (CLL) has been revolutionized in recent years, however CLL is still incurable, and the leukemic cells often develop drug resistance. Previous studies show that the interaction of CLL cells with the bone marrow (BM) microenvironment promotes spontaneous and drug induced survival. But the nature of this interaction is still in need of a full understanding. To pursue this we discerned RNA profiles using RNA-seq in paired CLL cells isolated from untreated blood and BM (n=6) and in untreated patients CLL cells (n=4) cultured alone or cocultured with BM stromal cells (BMSCs). CLL cells from blood/BM and CLL cells cultured alone or with BMSCs were examined by Western blot (WB), untargeted metabolomics (LCMS+GCMS) and preclinical drug sensitivity assays. We found upregulation of 232 genes in CLL cells from BM vs the paired blood and 917 genes in cocultured CLL cells compared to CLL cells cultured alone (p&lt0.05, fold change &gt1.5). Here we detected only 13 genes that overlap between BM and cocultured CLL cells. When we analyzed the expression of these 13 genes in blood CLL cells from a cohort of 162 untreated patients by RNA-seq we found a positive relationship between 4 (PNP, C16orf54, MOB3A, CDK2AP2) with CLL patient clinical outcome including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [multivariable Cox proportional hazards models, p&lt0.05]. Out of the 4 genes, purine nucleoside phosphorylase (PNP), an enzyme in the purine salvage pathway, showed the most significant association for patient’s OS, PFS, and TTFT. Metabolomic profiling indicated an increased level of purine salvage pathway metabolites adenosine, inosine, and hypoxanthine in cocultured CLL cells. WB analysis using blood CLL cells from untreated patients showed a variable PNP protein expression (high, low/no PNP) and induction in PNP protein levels were found in blood CLL cells expressing low/no PNP only when in contact with BM/BMSCs. But PNP inhibitor forodesine did not show any significant killing in high vs low/no PNP group. When we treated CLL cells from untreated patients with Bcl2 inhibitors venetoclax, S55746 and LP-118 (Bcl-2/Bcl-xl inhibitor, Newave), CLL cells showed increasing sensitivity to the Bcl2 inhibitors associated with their PNP expression (high&gtlow&gtno PNP). But a covalent and non-covalent BTKi LP-168 (Newave) cultured with CLL cells showed no difference in drug sensitivity associated with CLL PNP levels. In contrast CLL cells when treated with both venetoclax and LP-168, showed more drug sensitivity in the high PNP expressing CLL cells vs low/no PNP. These results indicate that active purine metabolism in CLL cells can contribute to differential novel agent drug responses of CLL patients. Future studies to understand the exact mechanism of how PNP relates to this differential drug sensitivity should be instructive in regard to alternate maneuvers to treat CLL. Citation Format: Sutapa Sinha, Weiguo Han, Zhiquan Wang, 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. Role of purine metabolism in CLL cell pathobiology and CLL disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 297.