Chronic Lymphocytic Leukemia Samples Research Articles

Abstract 4300: Large-scale, low-cost, and accurate measurement of cancer evolutionary dynamics from clinical patient samples

Abstract Introduction: Cancer is a dynamic evolutionary process; thus, the evolutionary history of a cancer should strongly influence its future trajectory and so the clinical outcome of patients. However, accurately assessing an individual cancer’s evolutionary history in a scalable and clinically relevant manner is currently an open problem. To address this, we introduce a novel methodology (EVOFLUx) to quantitively infer the history of a cancer from single time-point bulk samples using inexpensive, standard methylation arrays. Description of Methodology: We recently identified fluctuating CpGs (fCpGs), which are CpGs that neutrally and stochastically change methylation state over time in individual cells, uniquely barcoding cells and thus enabling high temporal-resolution lineage tracing approaches. In a bulk population of cancer cells, the distribution of fCpG methylation is determined by the evolution of the population and thus encode a cancer’s past clonal dynamics. Here, we have developed a general computational approach to model how the patterns of fCpGs vary according to the evolutionary history of a growing cancer, and a Bayesian inference method to infer these parameters from data. Results We applied EVOFLUx to quantify the evolutionary dynamics of 1,976 lymphoid cancers, finding that the tumor growth rates, malignancy age and epimutation rates varied by orders of magnitude across disease types. In addition, EVOFLUx allowed us to test for the presence of ongoing subclonal selection and validated our inference with matched whole exome sequencing data. Alongside characterizing the evolutionary dynamics within single bulk samples, fCpGs also allow for the inference of the phylogenetic relationships between longitudinal samples, which we validated with whole genome sequencing. Within cancer types, more aggressive subtypes typically had elevated growth rates. In our clinically annotated cohort of chronic lymphocytic leukemia (CLL) samples, we found that patient-specific evolutionary dynamics are strongly associated with outcome. Conclusions: Hence, we present a powerful new method that uses widely available, low-cost bulk DNA methylation data to precisely measures cancer evolutionary dynamics in patient samples with clinical implications. Citation Format: Calum Gabbutt, Martí Duran-Ferrer, Heather Grant, Diego Mallo, Ferran Nadeu, Jacob Househam, Neus Villamor, Olga Krali, Jessica Nordlund, Thorsten Zenz, Elias Campo, Armando Lopez-Guillermo, Jude Fitzgibbon, Chris P. Barnes, Darryl Shibata, José I. Martin-Subero, Trevor A. Graham. Large-scale, low-cost, and accurate measurement of cancer evolutionary dynamics from clinical patient samples [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 4300.

Investigating the potential of single-cell DNA methylation data to detect allele-specific methylation and imprinting

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p<8.4×10-9), and 32,862 CpG sites exhibited ASM in CLL samples (p<8.5×10-9). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

Abstract P01: Impact of Germline and Somatic ATM Variants in Chronic Lymphocytic Leukemia (CLL): Clinical Implications and Response to PARP Inhibition

Abstract Background: Next-generation sequencing has revealed numerous Variants of Unknown Significance (VUS) in the ATM gene, important in DNA repair and chronic lymphocytic leukemia (CLL) pathogenesis. This study evaluates the effect of rare ATM germline variants, including VUS, on features of CLL, ATM kinase activity, and response to PARP inhibition. Methods:ATM variants were detected in 885 CLL patients (327 DFCI; 276 German CLL Study Group; 282 ICGC) using DeepVariant, and compared to 5310 ancestry-matched controls. Concurrently, 278 CLL patients with ATM aberrations from clinical sequencing were retrospectively analyzed, annotated by population allele frequency (JCO 2023, Lampson et al.). ATM kinase activity was measured by ATM and KAP1 phosphorylation following gamma-irradiation in primary CLL samples. The impact of talazoparib, a PARP inhibitor, on ATM aberrant and wild-type CLL cell proliferation was also assessed. Results: Among the 885 CLL cases, 130 ATM germline variants were identified by DeepVariant, including 87 missense variants. The top 5 most frequent rare germline missense variants with high odds ratio for association with CLL were ATM p.L2332P (AF cases vs controls, 0.328% vs 0.0188%, OR 17.46), p.L2307F (OR 16.88), p.F763L (OR 18.89), p.Y1442H (OR 35.39) and p.S99G (OR 16.28). In our retrospective clinical cohort, we identified 294 ATM variants with 146 unique variants. Our previously proposed algorithm for classifying germline vs somatic identified 74 germline, with 70 missense, and 72 somatic. Phosphorylation of ATM and KAP1 (pATM, pKAP1) in response to IR was reduced in 13 patients assessed with germline ATM variants (9 rare and 4 non-rare; ATM variant alone group) compared to those without any somatic or germline ATM aberrancy (WT group) (P&amp;lt;0.01). Phosphorylation levels were also significantly lower in 17 patients with rare germline ATM variants with concurrent del(11q) (del11q + ATM germline variant group) compared to 15 with del(11q) alone (del 11q group) (P&amp;lt;0.01). We obtained similar results from Western blot analysis. To assess the sensitivity of ATM deficient cells to talazoparib, we compared proliferation inhibition across groups cocultured with talazoparib for 14 days. The del11q with ATM-somatic group exhibited significantly greater sensitivity to talazoparib at lower dosages compared to ATM WT (talazoparib 0.1 μM, P&amp;lt;0.05; 0.5μM, P=0.098). The monoallelic ATM deficient groups (del11q alone or ATM-germline alone) tended to have lower proliferation, but not significantly. Higher concentrations of talazoparib (2.5μM) strongly inhibited proliferation of all CLLs including WT. Conclusion: This study highlights the prevalence of rare ATM germline variants in CLL and their impact on ATM function in CLL. Our findings also suggest that these variants affect the DNA damage response in primary CLL cells and that ATM biallelic deficiency influences sensitivity to PARP inhibitors. Our evidence could lead to more personalized therapeutic strategies for CLL patients with distinct ATM aberrancies. Citation Format: Kiyomi Mashima, Nicholas Moore, Mariia Mikhaleva, Anna Petráčková, Samantha Shupe, Stacey M Fernandes, Amaro Taylor-Weiner, Riaz Gillani, Hoyin Chu, Seunghun Han, Sabrina Camp, Eric Kofman, Gad Getz, Catherine J. Wu, Svitlana Tyekucheva, Matthew S Davids, Eliezer Mendel Van Allen, Saud AlDubayan, Jennifer R Brown. Impact of Germline and Somatic ATM Variants in Chronic Lymphocytic Leukemia (CLL): Clinical Implications and Response to PARP Inhibition [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P01.

Analysis of Primary Chronic Lymphocytic Leukemia Cells' Signaling Pathways.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

T Cell Dysfunction and Exhaustion in Patients with CLL: The Impact of Long Term Ibrutinib Treatment

Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with defects in the immune system and is dependent on the microenvironmental niche for survival. Ibrutinib, the first irreversible inhibitor of Bruton's tyrosine kinase targets B-cell receptor signaling to kill tumor cells. In addition, ibrutinib also appears to regulate the tumor microenvironment and T-cell immunity, but this mechanism unknown. Increased expression of inhibitory receptors, and functional defects such as reduced proliferative capacity and cytotoxicity are common in T cells from patients with CLL. The effect of ibrutinib on T-cell immunity has not been extensively studied in the setting of long term ibrutinib treatment. Therefore, we investigated the phenotype and function of CD4+ and CD8+ T-cells from patients with CLL treated with ibrutinib, treatment naïve CLL and age-matched healthy controls. Methods: CLL patient samples were obtained from treatment naïve patients at diagnosis, and at the 3- and 5-year timepoints. Samples from patients on continuous ibrutinib who had not relapsed at the 3- and 5-year timepoints; and pre-ibrutinib were obtained as well. Age matched healthy donor-derived peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. PBMCs from CLL samples and healthy donors were labeled with CellTrace Violet and treated with anti-CD3 and anti-CD28 (ThermoFisher) for 7 days. T-cells were stimulated to identify proliferation capacity and to identify immune profiles. Results: Efficient T-cell activation and proliferation requires multiple signals such as T-cell receptor (TCR) stimulation, a costimulatory signal, and cytokine production. We analyzed the proliferation of T cells in-vitro upon stimulation with anti-CD3/CD28 beads using CellTrace Violet on PBMCs and found that this stimulation resulted in increased expansion of CD4+ T cells and CD8+ T cells in all the samples. We also found significant difference in the proliferation between untreated and treated CLL samples. Interestingly, proliferating T cells had different expression pattern of the exhaustion markers and inhibitory molecules such as TIM3, TIGIT, LAG3, CD160, KLRG1, CD244 and PD-1 at different phases of the disease. Increased expression of CD160, CD244, and PD1 are the key features of T-cell exhaustion. We found significant increase in the percentage of T cells from CLL patients expressing CD244, CD160 and PD1 compared to healthy donor controls but not between naïve and ibrutinib treated samples. Long term ibrutinib treatment in 3yr and 5yr samples does not change the exhaustion state in patients. We also found T-cells from ibrutinib treated samples express higher levels of CD57 which is known as a marker of senescence (Figure 1A). The phenotypes of untreated CLL samples are CCR7+CD45RA- central memory T-cells (35%) and CCR7+CD45RA+ naïve T cells (25%) (p&amp;lt;0.05) after stimulation in-vitro on day 3. In the same setting, ibrutinib treated samples had higher levels of effector memory T-cells (p&amp;lt;0.05) (80%) compared to untreated CLL samples (Figure 1B). These results suggest that the phenotype has changed to central memory phenotype in untreated samples. Functional T-cell exhaustion in CLL also leads to failure in cytokine production. Therefore, we measured different cytokine levels after stimulation in all the patient samples and in healthy controls. We found significant increase in the levels of inflammatory cytokines such as IFN-γ (p&amp;lt;0.05) and TNF-α (p&amp;lt;0.05) in long term ibrutinib treated samples compared to naïve CLL samples and healthy controls. Conclusion: Several studies have showed that T-cell counts are increased after treatment with ibrutinib in patients with CLL but not in extended therapy. Our findings suggest that long term ibrutinib treatment does increase T-cell number and proliferative capacity in CLL. The proliferating T cells express higher levels of exhaustion markers and inhibitory molecules in-vitro making them dysfunctional. However, these T-cells still maintained good proliferative potential, on par to healthy donors and better than untreated patients which can be applied in expanding improved T cell grafts for various types of adoptive T cell therapy, such as TCR gene therapy, and CAR therapy. Also better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells.

NK Cell Dysfunction in CLL Is Mediated through SHP-1 Signaling and Is Associated with Poor Prognosis

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, presenting with a highly variable clinical course, outcome and response to therapy. This variability has been linked to a complex tumor microenvironment and genetic/epigenetic modifications that lead to immunosuppression. Indeed, the clinical hallmark of this disease is the susceptibility to infections and secondary malignancies. While T cell dysfunction in CLL has been well described, conflicting information exists on NK cell changes in CLL and their role in CLL immunosurveillance. Here, we analyzed samples from 75 untreated (or completed treatment &amp;gt;2 years ago) CLL patients and 30 healthy controls. We discovered a substantial variation in NK cell function compared with healthy controls, which we further classified into hyperfunctional, normofunctional and dysfunctional based on their ability to degranulate, and produce IFN- γ in response to K562 targets ( Figure 1). NK cells from ‘dysfunctional’ CLL samples also displayed impaired cytotoxicity against K562 targets, weaker synapse formation and Syk/Zap70 signaling in response to antibody-dependent cellular cytotoxicity (ADCC) compared to those from ‘functional’ samples. The NK functional status was strongly associated with multiple prognostic markers, including cytogenetics, IgVH mutation status, ZAP-70 expression and disease stage, with patients with dysfunctional NK cells having significantly higher rates of secondary malignancies and viral infections ( Table 1). Using multispectral flow cytometry we discovered high phenotypic heterogeneity with unique NK cell clusters present in the dysfunctional samples. Those clusters express a pattern of activation-induced exhaustion, with upregulation of multiple checkpoint molecules such as PD-1, TIGIT, LAG-3, KLRG-1, TIM-3 but not evidence of terminal differentiation (CD57 negative). Those clusters also showed concomitant expression of activating molecules such as NKG2D and DNAM-1, suggesting reversibility of their dysfunctional status. We subsequently found significantly higher (p&amp;lt;0.0001) constitutive phosphorylation of SHP-1 protein, a phosphatase downstream of multiple inhibitory NK cell receptors, in NK cells from dysfunctional samples. Treatment with a SHP-1 inhibitor NSC-87877 (EMD Millipore) or use of siRNA targeting SHP-1 (ThermoFisher scientific), resulted in reversal of NK dysfunction with full restoration of their functional capacity. Taken together, our data suggest that NK cells functional status plays an important role in CLL disease progression and secondary complications. The novel mechanism of NK cell immune dysfunction in CLL described here has the potential to improve outcomes with novel immunotherapies such as chimeric antigen receptors, checkpoint inhibitors and bi-specific antibody therapy that have shown disappointing results in CLL to date when compared with other B cell malignancies.

Pre-Clinical Study on the Dual BCL2/BCL-XL Inhibitor AZD0466 for the Treatment of Chronic Lymphocytic Leukemia

Targeting BCL2 with venetoclax has proven successful for treatment of chronic lymphocytic leukemia (CLL), but resistance develops in a subset of patients. Among other mechanisms, mutations in BCL2 and increased BCL-XL expression confer venetoclax refractoriness in patient subgroups. It is therefore important to identify alternative treatment options targeting BCL-XL and BCL2 for combating venetoclax resistance. AZD4320 was developed as a dual BCL-2/BCL-XL inhibitor. AZD0466 is a dendrimer-drug conjugate of AZD4320, designed to optimize drug release and improve drug tolerability. Hence, we used the nano drug AZD0466 for in vivo experiments and AZD4320 for in vitro and ex vivo experiments. Firstly, we studied the efficacy of AZD0466 in vivo in the Eµ-TCL1 murine CLL adoptive transfer model. Due to prevalence of primary resistance, murine tumors with the best ex vivo response to AZD4320 were selected and 10 million cells were transferred by i.v. in syngeneic recipient mice. Upon reaching a tumor load of 10% CD19+ CD5+ cells in peripheral blood, mice were randomized and treated with 30mg/kg ibrutinib in drinking water, 34mg/kg or 70mg/kg of AZD0466 once weekly (QW) by i.v, or the vehicle. Treatments were continued until reaching a predefined humane endpoint. AZD0466 at 70mg/kg led to a significant increase in survival (median 72 days vs. 38 days, P=0.0009) similar to ibrutinib (67 days, P=0.0026) when compared to vehicle (Fig. 1). Analysis of blood parameters at euthanasia did not reveal significant difference in platelet counts between AZD0466, ibrutinib and vehicle treatments. To evaluate if combination treatment of AZD0466 with BTK inhibitors would improve efficacy, we transplanted murine Eµ-TCL1 tumors into syngeneic recipient mice and randomized them for treatment with vehicle, ibrutinib (30mg/kg in drinking water), acalabrutinib (25mg/kg, p.o. QD), AZD0466 (70mg/kg, i.v., QW) and combination of AZD0466 with ibrutinib or acalabrutinib. AZD0466 and vehicle treatments showed similar platelet and RBC counts measured 2 or 3 days after the 2 nd and 4 th dosing but, a significant increase in platelet and RBC counts were observed with the combination treatments. A significant decrease in WBC count after 2 weeks, and in bone marrow tumor load after 4 weeks was observed with combined AZD0466 and acalabrutinib treatment compared to the single drugs. Though spleen weights did not differ after 4 weeks between vehicle and AZD0466 monotherapy, a strong decrease in spleen weight and tumor cell number in spleen was observed with the combination of AZD0466 with ibrutinib or acalabrutinib compared to vehicle. To validate the findings in human CLL, we performed dynamic BH3 profiling on primary CLL samples with mutated (N=5) and unmutated IGHV (N=7). Exposure of CLL cells to 1µM acalabrutinib for 24 hours significantly increased BCL2 dependency in all CLL samples with unmutated IGHV and in a subset of mutated IGHV samples. These tumors also showed an increased response to venetoclax and AZD4320 upon exposure to acalabrutinib. To study the efficacy of the dual BCL2/BCL-XL inhibitor in the context of venetoclax resistance, we stably expressed the BCL2 mutations A113G, D103E, D103V, D103Y, G101V, R129L, V156D and wildtype (WT) in the venetoclax sensitive mantle cell lymphoma cell line MAVER-1. Response to AZD4320 or venetoclax was analyzed using Annexin V/7AAD staining and flow cytometry after 48 hours of treatment. The BCL2 mutants showed varying degrees of resistance to venetoclax. MAVER-1 expressing WT had an IC50 of 17.7nM while D103E, D103V, D103Y, G101V showed the strongest resistance to venetoclax with 5, 13.8, 4.5 and 7.3 fold increase in IC50 compared to WT. Of note, compared to WT (IC50 43.9nM), D103E, D103V and D103Y were sensitive to AZD4320 with a fold change in IC50 of 0.6, 1.8 and 1.3, respectively. G101V mutant cells showed a 5 fold increase in IC50 to AZD4320 compared to WT (Fig. 2). Moreover, AZD4320 was highly efficacious in MAVER-1 and MINO cell line models where resistance to venetoclax mediated by BCL-XL upregulation was modelled by an in vitro dose escalation method. In summary, our pre-clinical study shows that the dual BCL2/BCL-XL inhibitor could represent an important treatment option for venetoclax resistance mediated by specific BCL2 mutations or BCL-XL upregulation and that its efficacy could be further improved upon combination treatment with BTKi.

B-Cell Receptor Function and Gene Expression Profiling at Single-Cell Level across the Spectrum from Normal to Neoplastic CD5-Positive B-Cell Compartments in Humans

Antigen-independent, autonomous signalling of the B-cell receptor (BCR) is an oncogenic driver common to all cases and subtypes of chronic lymphocytic leukemia (CLL). We have recently shown that the dominant clonal population of low-count (&amp;lt;500 cells/µL) monoclonal B lymphocytosis (MBL) with CD5 +CD20 lowCD79B low CLL phenotype identified prospectively in healthy siblings of CLL patients also exhibit autonomous BCR signalling, albeit with lower signalling strength than CLL BCR (Quinten et al., Haematologica 2023). To gain further insight into the mechanisms of clonal expansion within the CD5 + B-cell compartment as the possible origin of CLL, we performed single-cell gene expression profiling and exemplary analyses of BCR function in normal CD5 + B cells (CD20 hiCD79B hi) and in CLL-phenotype B cells (CD5 +CD20 lowCD79B low) of healthy siblings of CLL patients in comparison to CLL and high-count MBL cells. Screening of blood samples of 16 sibling stem cell donors of CLL patients by flow cytometry revealed the presence of CD5 +CD20 lowCD79B low B cells at low counts in 5 donors. These cells and normal CD5 +CD20 hiCD79B hi B cells were sorted in parallel, pooled at equal ratios, and analyzed together with CLL B cells from their sibling stem cell recipients and 2 unrelated high-count MBL B cells on two 10X Genomics chips for single-cell 5‘ gene expression and BCR sequencing. Data deconvolution per individual was performed based on expressed SNPs and on the individuals’ HLA types. In a combined 2-dimensional t-SNE plot based on the 2000 most variably expressed genes, the normal CD5 +CD20 hiCD79B hi B cells from all 5 donors clustered distinctly from CLL-phenotype cells from all sources (dotted lines, Figure) and indeed showed significantly higher expression of CD20 and CD79B. Gene set enrichment analysis revealed significant upregulation of the hallmark oxidative phosphorylation gene set in all CLL samples compared to non-CLL B-cell populations. BCR analysis demonstrated clonal dominance of &amp;gt;99% for both Ig heavy and light chains in the 5 CLL samples. In accordance with the indication for allogeneic stem cell transplantation, these CLL BCR were unmutated. In marked contrast, normal CD5 +CD20 hiCD79B hi B cells from the healthy SC donors were also unmutated but completely polyclonal, i.e. no identical BCR was detected in any two cells of this compartment. In 4/5 donors, the CLL-phenotype CD5 +CD20 lowCD79B low compartment contained multiple clonal expansions of various degrees. In one high-count MBL, 17 other B-cell clones with various degrees of low-level expansion coexisted with the &amp;gt;90% dominant MBL clone. BCR from the most dominant and one or more of the less expanded CD5 +CD20 lowCD79B low clones of the healthy donors were tested for autonomous signaling as quantified by BCR-induced calcium flux without experimental cross-linking in murine TKO pre-B cells and compared to BCR from nonexpanded CD5 +CD20 hiCD79B hi cells and from their CLL siblings. Autonomous BCR signaling strength was calculated from the fraction of cells with spontaneous calcium flux above test background and calibrated against calcium flux after BCR crosslinking. CLL and high-count MBL BCR had robust median autonomous BCR signaling of 0.326. Expanded CD5 +CD20 lowCD79B low clones of the healthy donors showed significantly weaker BCR signalling strength of 0.163 (p&amp;lt;0.01; Scatter Plot). BCR signalling in nonexpanded CD5 + B cells appeared weaker than in expanded clones but was clearly above the background of BCR from mature B cells isolated from lymph node biopsies expressing a mutated IgG. In conclusion, the low-count CD5 +CD20 lowCD79B low CLL-phenotype B-cell compartment in healthy adults is composed of multiple, predominantly mutated B-cell clones. Therefore, the term “monoclonal B lymphocytosis” is highly misleading for these B-cell populations and should be avoided. Normal CD5 +CD20 hiCD79B hi B cells are exclusively unexpanded and express unmutated BCR, indicating a naïve status. Unexpectedly, these nonexpanded CD5 + B cells express BCR with relatively weak but quantifiable autonomous signalling. Globally, autonomous BCR signalling strength across the CD5 + B-cell compartment appears to be correlated to the degree of clonal expansion. Fully malignant CLL cells overexpress genes of the oxidative phosphorylation pathway, presumably to meet increased energy demand from alternative sources.

The Gene Expression Landscape of BTK Inhibitor Treatment in Chronic Lymphocytic Leukemia Exposed By Shallow Depth RNA Sequencing

Efforts to understand chronic lymphocytic leukemia (CLL) subgroups have focused on multi-omics profiling of patient cohorts (in steady-state) and linking biomarkers to responses. Here, we aim to combine ex-vivo drug screening and gene expression profiling (GEP) in primary CLL samples to dissect disease biology based on the observed phenotype as a qualitative and quantitative function of steady-state features and drug perturbation effects. As most differential gene expression changes in CLL are retrieved from highly expressed genes, we hypothesized that a relevant set of genes could still be reliably captured by reduced sequencing coverage. We profiled five samples with traditional whole transcript TruSeq and 3'-end QuantSeq (Moll et al., Nat Methods, 2014) library preparation with deep and shallow depth sequencing, respectively. We obtained 43.48-72.68 million mappable reads with TruSeq and 2.24-3.78 million with QuantSeq. Although the read coverage was reduced, QuantSeq was able to recover 71.17 ± 0.87% of all genes that were detected by TruSeq with reproducible quantification ( R &amp;gt; 0.85, P &amp;lt; 0.001 for all samples). Shallow sequencing of CLL samples reliably profiled intermediate to highly abundant genes with reduced sensitivity only for lowly expressed genes. We then selected 105 representative CLL patient samples from a well-characterized cohort, treated them ex-vivo with 100 nM ibrutinib for 48h and performed GEP using shallow depth QuantSeq. Sample viability was measured by flow cytometry before and after perturbation. Library preparation and sequencing was successful for 91% of samples. With a median coverage of 2.19 million mappable reads per sample, the principal components (PC) of transcriptional variation in DMSO-treated samples included IGHV (12.0%), methylation subgroup and trisomy 12 status (7.1%). These data show that the screening method reliably captured the transcriptional features of CLL at steady-state and thus support the use of shallow depth RNA sequencing. We were interested in the landscape of transcriptional change after perturbation of the B-cell receptor (BCR) pathway. Samples from the same patients clustered together and spread along a gradient of sample viability in the first PC. After correlation of gene counts from DMSO-treated samples with sample viability and subsequent gene set enrichment analysis, we found activation of the canonical apoptosis pathway via NFκB-mediated TNF-α signaling and cell stress to be associated with this signature of cell death. BTKi-treated samples showed the same signature of spontaneous apoptosis and additionally strong suppression of MYC and OXPHOS activity. To identify the ibrutinib-regulated transcriptional signature, we performed differential gene expression analysis between ibrutinib and DMSO. BTKi treatment induced 904 differentially expressed genes (DEGs) ( FDR &amp;lt; 0.005). These genes were negatively enriched in the hallmarks allograft rejection, MYC targets, TNF-α signaling via NFκB and KEGG pathways associated with BCR signaling, and immune cell response. Top DEGs included e. g. FCRL5, PTPN6, FILIP1L, LRMP and LAPTM5 ( FDR &amp;lt; 0.001) which were also significantly downregulated after in-vivo ibrutinib exposure (Rendeiro et al. Nat Commun, 2020). Inhibition of the BCR induces apoptosis in CLL in an IGHV status-dependent manner. Samples with unmutated IGHV (UM-CLL) were more sensitive to BTKi compared to samples with mutated IGHV (M-CLL) (83.1 vs. 92.3% relative viability, P &amp;lt; 0.001). On gene expression level, we observed more DEGs within UM-CLL (387) compared to M-CLL (182) ( FDR &amp;lt; 0.005). Response to BTKi was largely similar although the effect size in M-CLL tended to be smaller. To identify BCR-dependent genes that were significantly modulated by IGHV, we tested for the interaction between BTKi treatment and IGHV status in the differential expression models. We found the effects on the expression of CA12, FIG RPL5 and P2RY11 to be different upon BTK inhibition between UM- and M-CLL ( FDR &amp;lt; 0.1). In conclusion, we establish shallow depth sequencing in CLL and show it can be used to reliably capture disease biology and drug effects. By treating patient samples with ibrutinib, we show that abrogating BTK strongly suppresses BCR-dependent processes which are largely independent of IGHV status. By adding more pathway perturbations, this approach could be used for further functional dissection of CLL biology.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with &amp;gt;260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

MS-553 Therapy Inhibits BCR Signaling and Increases Survival Dependence on BCL-2 in BTK-Inhibitor Resistant CLL

B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL. We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

A Phase 1 Study of Adding Pitavastatin to Venetoclax-Based Therapy in AML and CLL/SLL

Statins (HMG-CoA-reductase inhibitors) are drugs used widely for the treatment of high cholesterol and are generally well-tolerated. There has been increasing interest in repurposing statins for oncology based on the recognition that many cancer cells are “addicted” to the mevalonate biosynthetic pathway (Iannelli et al, 2017; Juarez and Fruman 2021). We have shown that statins increase expression of the pro-apoptotic BCL2 family member PUMA (Lee at al, 2018). We have strong pre-clinical evidence that statins improve responses to the BCL2 inhibitor venetoclax (VEN) in cell line models and ex vivo primary patient specimens from patients with both chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) (Lee et al, 2018). Additionally, retrospective review of patients enrolled into the early clinical trials of VEN in CLL revealed improved responses to VEN (complete remission [CR] and progression-free survival) in the patients who were concurrently on a statin medication (Lee et al, 2018). These retrospective analyses may underestimate the potential of statins for leukemia therapy, as the statin drugs most commonly prescribed for hypercholesterolemia are not pharmacologically optimized for oncology (Abdullah et al., 2018). We developed a phase 1 study of adding pitavastatin (PIT) to VEN-based therapy in patients with AML and CLL/small lymphocytic lymphoma (SLL) (NCT04512105). AML patients were eligible if receiving induction therapy with azacitidine (AZA) and VEN per standard of care. CLL/SLL patients could receive either VEN with obinutuzumab (O) or rituximab. A 3+3 design was employed. Dose level 1 was PIT 2 mg daily, and dose level 2 (DL2) was 4 mg daily. These doses are consistent with the doses used for the treatment of hypercholesterolemia. Those enrolling at DL2 had a creatinine clearance of 60 mL/min or higher based on the prescribing information for pitavastatin. Patients already on statins for the treatment for hypercholesterolemia were allowed; these patients stopped their prescribed statin for 72 hours prior to initiation of PIT. PIT started after patients completed VEN ramp-up and had been on a stable VEN dose for at least 5 days. Primary objectives were assessing safety and tolerability and determining the recommended phase 2 dose of PIT in combination with VEN-based therapies. Secondary objectives included CR rates. Exploratory objectives included performing BH3 profiling on pretreatment and post-treatment blood samples to assess whether add-on treatment with PIT increases apoptotic priming, as seen in preclinical models (Lee et al, 2018). 14 patients signed informed consent. 6 were deemed ineligible. 2 withdrew consent prior to starting PIT therapy. 6 patients received PIT on study. 2 patients had AML and received AZA+VEN. 2 patients had CLL, and 2 patients had SLL. The CLL/SLL patients all received VEN+O. Grade 1 myalgia was reported in one subject on the 4mg PIT dose. 1 patient experienced a grade 1 increase in AST on the 4mg dose. Another patient had grade 1 elevation in bilirubin and alkaline phosphatase at the 4 mg dose. Grade 3-4 toxicities included leukopenia, neutropenia (1 case of febrile neutropenia, grade 3), anemia, and thrombocytopenia. Cycle 1 was defined as the dose-limiting toxicity (DLT) period, and no hematologic toxicities occurred during the DLT period. 1 patient had grade 3 pancreatitis, but this was felt to be unrelated to therapy or disease (also outside the DLT period). No grade 5 toxicities occurred while on study therapy. Toxicities were overall felt to be similar to patients receiving VEN-based therapy in standard clinical practice. All evaluable patients (n=5) achieved clinical CR. 1 patient with SLL is awaiting re-staging at the time of this abstract. Correlative studies are ongoing. In 2 patient peripheral blood samples analyzed, the % blasts (for an AML sample) and % CD5+ B-cells (for a CLL sample) were reduced 24 hours after PIT dosing compared to prior to any treatment and before PIT/after VEN ramp-up. Based on the tolerability of PIT and encouraging clinical outcomes, a phase 2 study is being planned to better assess the efficacy of adding PIT to VEN-based therapies in AML and CLL/SLL.

Characterizing ATM Aberrations in Chronic Lymphocytic Leukemia (CLL): Prognostic Implications and Sensitivity to PARP Inhibition

Introduction ATM mutations (muts) frequently occur in CLL, yet their influence on ATM protein function and DNA break repair is poorly elucidated. Our prior study demonstrated functional hypomorphism of the rare germline variant L2307F in ATM and a higher occurrence of 11q deletions (del(11q)) with ATM germline variants in CLL (Lampson et al, JCO 2023). However, the role of other ATM germline variants in association with del(11q) or somatic muts remains unknown. Here, we analyzed the characteristics of CLL patients (pts) with ATM aberrancies and evaluated ATM function in primary CLL cells. Methods We retrospectively analyzed 278 CLL pts with ATM aberrancy, evaluated by clinical targeted sequencing from 2014 to 2019, as per our recent study (Lampson et al, JCO 2023). ATM, KAP1, and H2AX phosphorylation levels were examined after etoposide or irradiation (IR) exposure, by western blot or flow cytometry, in primary CLL cells with or without ATM aberrancy. We assessed the inhibitory effect of talazoparib, a PARP inhibitor, on CLL proliferation over 14 days using flow cytometry and Cell Trace Violet dye. Results We identified 294 ATM variants, 146 unique (germline N=74, somatic N=72). In CLL pts, 58 had dual ATM allele loss (biallelic group), including del(11q) together with only germline ATM variant(s) (N=29), only somatic mut(s) (N=18), or both (N= 11). 168 pts had ATM muts or variants without del(11q), including only germline (N=137), somatic (N=19), or both (N=12) ( ATM group), and 52 pts had del(11q) alone (11q group). Complex karyotype increased with ATM allele deficiency, particularly with 5+ abnormalities (complex karyotype 5+, biallelic vs 11q vs ATM: 22.6% vs 10.6% vs 5.8%, P=0.002). Unmutated IGHV was significantly higher in the groups with del(11q) versus ATM alone (biallelic vs 11q vs ATM: 85.1% vs 87.8% vs 53.9%, p&amp;lt;0.001). Interestingly, when we compared IGHV status in the ATM alone group with or without somatic ATM muts, unmutated IGHV significantly increased in those with somatic muts ( ATM-somatic vs ATM-germline alone, 81.8% vs 43.6%, p&amp;lt;0.001), and karyotypic complexity tended to be higher in the somatic ATM mut group (P=0.077). This might suggest that pts with ATM somatic muts have clinical characteristics and pathogenicity more similar to del(11q) as compared to those with germline muts. SF3B1 muts were more common in those with del(11q) (biallelic vs 11q vs ATM: 20.7% vs 26.9% vs 12.5%, P=0.036). For functional evaluation, we compared the five groups of primary CLL samples: del(11q) with somatic ATM mut with/without germline (11q+ ATM-S), del(11q) alone with no ATM muts (11q), somatic ATM muts without del(11q) with/without germline ( ATM-S), germline ATM muts alone ( ATM-G), and no ATM aberrancies (wild type, WT). Western blot demonstrated significantly lower phosphorylated protein levels (pATM, pKAP1, γH2AX) in response to DNA damage with etoposide in ATM aberrancy groups compared to WT (WT vs 11q, vs ATM-S, vs 11q+ ATM-S, all P&amp;lt;0.001 except for WT vs 11q in γH2AX, P&amp;lt;0.05) (Figure 1A). Flow cytometry also showed significantly lower phosphorylation levels of ATM after IR in 11q, ATM-S and 11q+ ATM-S groups compared to WT, while ATM-G group didn't show significance. To assess ATM deficient cell sensitivity to talazoparib, we compared proliferation inhibition across groups cocultured with talazoparib for 14 days. The 11q+ ATM-S group exhibited significantly greater sensitivity to talazoparib at lower dosage compared to the WT (talazoparib 0.1 μM, P&amp;lt;0.05; 0.5 μM, P=0.098) (Figure 1B). The monoallelic ATM deficient groups (del11q and ATM-G) tended to have lower proliferation, but not significantly. Higher concentrations of talazoparib (2.5μM) strongly inhibited proliferation of all CLLs including WT. Conclusions Pts with ATM somatic aberrancy including del11q had worse associated prognostic factors including unmutated IGHV and complex karyotype. Somatic events showed greater functional impact than some germline variants. CLL cells with ATM aberrancies had ATM pathway dysfunction, and those with biallelic ATM aberrancies were more sensitive to the PARP inhibitor, talazoparib, than WT. This work could potentially lead to additional avenues for treatment based on differential sensitivities of CLLs with ATM aberrancy. Further studies are needed to characterize specific ATM germline variants with or without somatic ATM events.

Live Lymphoma and CLL Cells inside the Bone Marrow Fibroblasts: Implication in Residual Disease Persistence

Bruton tyrosine kinase inhibitors (BTKi) have generated remarkable clinical responses in patients with chronic lymphocytic leukemia (CLL) and other mature B-cell malignancies. However, deep, and durable response to BTK inhibition is uncommon. A significant number of patients with measurable residual disease relapse with resistance to therapies. Although mutational mechanisms responsible for resistance have been well described, it is not known what mechanism led to persistence of the minimal residual disease (MRD) in the first place. The tumor microenvironment (TME) contributes to drug resistance. Various roles for stroma participation have been described, however, none of these include the physical intracellular sheltering of tumor cells. Cell-in-cell (CIC) describes the microscopic observations of the embodiment of an intact cell by another whole cell ( Fig. 1A). Pathologists have reported this phenomenon sporadically in human fixed tissues for over a century. However, its biological, pathological, and clinical significance remains obscure. In the absence of systematic studies, understanding the significance of live CIC remains challenging. In this investigation, we studied primary human CLL samples using an ex vivo co-culture model system which mimics the CLL TME. In the TME system, CLL cells were cocultured with a primary bone marrow fibroblast line (BMF) in the presence of B-cell growth factors. The tumor cells survive for weeks, become larger, downregulate surface CXCR4. Phenotypically, tumor cells proliferate and divide into daughter cells. Collectively, these features recapitulate the behavior of lymph node-resident CLL cells. Using the TME system, we have demonstrated that modeled drug response correlates well with patient clinical response to BTK inhibitor ibrutinib and BCL2 inhibitor venetoclax. Interestingly, with confocal microscopy and 3D reconstruction, we observed that CLL cells placed in the model system were actively internalized by BMF in all 41 CLL cases studied. CLL cells were live and mobile. Moreover, one CLL cell was captured halfway in ( Fig. 1B). The number of internalized CLL cells was quantified and correlated with patients' clinical and pathological features. No correlations were found between CIC and widely used CLL prognostic indicators, including cytogenetic features and IGHV mutational status. However, we have found that patients who were on treatment for CLL at the time of sample collection had higher levels of CIC than those not treated. We hypothesized that live CIC may be an important mechanism contributing to residual disease persistence and drug resistance. To test the hypothesis, we exposed CLL cells in the TME model system to covalent and noncovalent BTKi including ibrutinib, zanubrutinib, and pirtobrutinib at clinically achievable concentrations. Our results showed that drug exposure drove CLL cells into BMF. This happened specifically to the BTK inhibitors as BCL2 inhibitor venetoclax was not observed to cause such an increase in CIC. These laboratory findings are consistent with the clinical observations that the rate of MRD negativity is much higher with venetoclax treatment than with BTKi. Additionally, CIC was observed in both follicular lymphoma (FL) cell line and primary FL cells suggesting the phenomenon is not limited to CLL. Further, we found that CIC was stimulated by several B-cell growth factors. The chemokine CXCL12-CXCR4 axis played a critical role in this process. Specifically, conditioned medium containing CXCL12 ligand promoted cell internalization, while plerixafor, a CXCR4 antagonist, largely prevented CIC from occurring. Furthermore, plerixafor blocks ibrutinib-driven CIC in cell lines as well as in primary CLL cells. Our study represents the first report implicating the CIC phenomenon in residual disease persistence using a cohort of patient-derived CLL cells assessed with 3D imaging and quantitative approach. These findings suggest that live CIC may be an important mechanism tumor cells use to avoid harmful drug insults, survive, persist, and later proliferate, eventually leading to clinical relapse. Targeting the CXCL12-CXCR4 axis may become a potential therapeutic strategy to minimize residual disease and future relapse. Fig.1. A. Images are reconstructed in 3D using Imaris v10.0. BMF cells are visualized as transparent red surfaces and CLL cells as green surfaces. B. One CLL cell was captured halfway in.

Aberrant Heterochromatin Silences Immune Response Genes in Chronic Lymphocytic Leukemia

Introduction: Recent studies have shown that clonal B cells derived from patients with chronic lymphocytic leukemia (CLL) have altered histone modification landscapes compared to B cells from healthy controls. We have also shown that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells, implicating the interaction between oncogenic signaling and epigenetic machinery in CLL (Wang et al., Blood Cancer J. 2022; Wang et al., Cancer Res (2023) 83 (7_Supplement): 4725). However, the majority of the chromatin studies in CLL concentrate on the active enhancers associated with oncogene expression; there is a very limited understanding of lost enhancers in CLL: 1) What is the function of lost enhancers; 2) How are these lost enhancers maintained in CLL; 3) Can these lost enhancers be detected at the precursor state of CLL, monoclonal B-cell lymphocytosis (MBL); 4) if this aberrant chromatin signature affected by CLL treatment. Methods: We systematically mapped the genome-wide histone modification profile (histone marks: H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, and H3K36me3) in 22 previously untreated CLL samples, 6 high count MBL samples, and peripheral blood B cells from 6 healthy donors by Cleavage Under Targets and Tagmentation (CUT&amp;Tag). We also analyzed B cells from 8 patients before and after one year of continuous Bruton tyrosine kinase inhibitor (BTKi) ibrutinib treatment. The genomic regions with a decrease in both H3K4me1 and H3K27ac enrichment (analyzed by Deseq2, fold change &amp;gt; 1, p &amp;lt; 0.01) were named lost enhancers. We used CRISPR-mediated epigenetic editing to study the role of gained H3K9me3 on EBF1 gene expression. Results: Compared to normal B cells, the lost enhancers (genomic regions defined by decreased enrichment of H3K4me1 and H3K27ac) in CLL B cells showed increased heterochromatin marks (H3K9me3 and H3K27me3) enrichment. Importantly, these enhancers are associated with genes downregulated in CLL and involved in the immune response, indicating the source of epigenetic impact on impaired immunity in CLL. We further found that this aberrant chromatin structure also exits in clonal B cells from the precursor stage of MBL, implicating the importance of epigenetic silencing in CLL evolution. In particular, we found the lost enhancer signature across the gene locus EBF1, an important transcription factor that defines B cell lineage and regulates normal B cell activation. Using CRISPR-mediated epigenetic editing, we demonstrated that the gain of H3K9me3 is sufficient to silence the EBF1 enhancer and its gene expression. Finally, we found that while BTKi ibrutinib treatment partially inhibits the CLL-gained enhancers in some patients, it failed to restore the lost enhancers in CLL B cells. Conclusion: In summary, we have elucidated an epigenetic silencing mechanism that may contribute to the loss of physiological function of normal B cells, which places the clonal B cells at a selection advantage during CLL evolution and increases the risk of serious infections in CLL and MBL ( Fig 1).

A-156 White Blood Cell Measurands, Flagging and Scatter Plot Patterns in Chronic Lymphocytic Leukemia

Abstract Background In this study, we use Alinity hq hematology analyzer to investigate the differences between white blood cell measurands of a healthy control population vs Chronic Lymphocytic Leukemia (CLL). A repeatability study was also conducted to determine the precision of the white blood cell parameter results on the Alinity hq. In addition, we used Alinity hq to identify unique flagging and patterns in the optical channels for CLL. Methods One hundred and fifteen peripheral blood samples of CLL were compared to a population of four hundred and fourteen healthy controls on the Alinity hq analyzer. Statistical differences in Alinity hq’s white blood cell measurands compared between the control population and CLL. For the repeatability study, five CLL samples (with sufficient volume) will be tested in 10 replicates for WBC measurands. These samples can be part of the 115 samples above or can be different samples. Comparison of scatterplots were also analyzed between CLL and control samples. Lastly, a peripheral smear was performed on all CLL samples. Results Statistical analysis of the means of the white and red cell parameters showed that in comparison to the means of the control population there was a significant statistical (P &amp;lt; 0.05) and clinical difference with CLL for the following parameters: absolute white blood cells (WBC) X 103/µL (43.6 CLL vs 7.41 control), percent neutrophils (%N) (18.25 CLL vs 53.71 control), percent lymphocytes (%L) (77.72 CLL vs 33.47 control), and percent monocytes (%M) (2.71 CLL vs 8.75 control). The WBC repeatability study results showed good concordance on the Alinity hq CBC + Diff + Retic test mode with the following percent coefficient of variance (%CV) results: absolute WBC X 103/µL (0.64, 5.31), percent neutrophils (%N) (1.32, 5.57), percent lymphocytes (%L) (0.20, 2.49), and percent monocytes (%M) (3.99, 26.39). As expected, the %CV range for monocytes is wide due to lymphocyte/variant lymphocyte population extending into the monocyte cluster. Evaluation of flagging showed 67 of the 115 (58.26%) CLL samples had a blast flag and 88 out of the 115 (76.52%) CLL samples had a variant lymphocyte flag. Review of the raw data from the optical channels shows a dominant and elongated lymphocyte cluster on the IAS, FL1 and PSS channels vs ALL for the CLL sample group compared to the control population. In the IAS and PSS vs ALL scatter plots in the CLL samples the lymphocyte cluster extends into the monocyte population triggering the blast flag. Peripheral smear review confirmed the presence of CLL (i.e., numerous smudge cells, mature-looking lymphocytes with characteristic condensed “soccer ball” pattern chromatin and scant cytoplasm) in the experimental group. Conclusion CLL is a cancer of the blood and bone marrow that predominately affects the quantity, and morphology of lymphocytes. Patients present with an abnormally high relative and variant lymphocyte populations. As such, there are a number of signs found in WBC measurands, flagging, and scatter plots on the Alinity hq that can be used as an early screening tool for CLL.

Effects of blocking CD24 and CD47 'don't eat me' signals in combination with rituximab in mantle-cell lymphoma and chronic lymphocytic leukaemia.

Mantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) with a poor prognosis, at high risk of relapse after conventional treatment. MCL-associated tumour microenvironment (TME) is characterized by M2-like tumour-associated macrophages (TAMs), able to interact with cancer cells, providing tumour survival and resistance to immuno-chemotherapy. Likewise, monocyte-derived nurse-like cells (NLCs) present M2-like profile and provide proliferation signals to chronic lymphocytic leukaemia (CLL), a B-cell malignancy sharing with MCL some biological and phenotypic features. Antibodies against TAMs targeted CD47, a 'don't eat me' signal (DEMs) able to quench phagocytosis by TAMs within TME, with clinical effectiveness when combined with Rituximab in pretreated NHL. Recently, CD24 was found as valid DEMs in solid cancer. Since CD24 is expressed during B-cell differentiation, we investigated and identified consistent CD24 in MCL, CLL and primary human samples. Phagocytosis increased when M2-like macrophages were co-cultured with cancer cells, particularly in the case of paired DEMs blockade (i.e. anti-CD24 + anti-CD47) combined with Rituximab. Similarly, unstimulated CLL patients-derived NLCs provided increased phagocytosis when DEMs blockade occurred. Since high levels of CD24 were associated with worse survival in both MCL and CLL, anti-CD24-induced phagocytosis could be considered for future clinical use, particularly in association with other agents such as Rituximab.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

T-cell dysfunction by pseudohypoxia and autocrine purinergic signaling in chronic lymphocytic leukemia

AbstractAcquired T-cell dysfunction is common in chronic B-cell malignancies. Given the strong connection between T-cell metabolism and function, we investigated metabolic alterations as the basis of T-cell dysfunction induced by malignant cells. Using B-cell malignant cell lines and human peripheral blood mononuclear cells, we first established a model that recapitulates major aspects of cancer-induced T-cell dysfunction. Cell lines derived from chronic lymphocytic leukemia (CLL) (PGA-1, CII, and Mec-1), but not from other B-cell malignancies, altered the T-cell metabolome by generating a pseudohypoxic state. T cells were retained in aerobic glycolysis and were not able to switch to oxidative phosphorylation (OXPHOS). Moreover, T cells produced immunosuppressive adenosine that negatively affected function by dampening the activation, which could be restored by the blocking of adenosine receptors. Subsequently, we uncovered a similar hypoxic-like signature in autologous T cells from primary CLL samples. Pseudohypoxia was reversible upon depletion of CLL cells ex vivo and, importantly, after the in vivo reduction of the leukemic burden with combination therapy (venetoclax and obinutuzumab), restoring T-cell function. In conclusion, we uncovered a pseudohypoxic program connected with T-cell dysfunction in CLL. Modulation of hypoxia and the purinergic pathway might contribute to therapeutic restoration of T-cell function.