Survival In Chronic Lymphocytic Leukemia Research Articles

Abstract 5842: An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia

Abstract Therapeutic inhibition of BCR signaling using Bruton tyrosine kinase inhibitors (BTKis) has remarkable efficacy in the treatment of chronic lymphocytic leukemia (CLL). However, the efficacy of BTKis is limited by the development of drug resistance, which can be mediated through mutations in genes encoding proteins of BCR signaling pathways. Approaches targeting the BCR mutant proteins have been limited by the large heterogeneity of these mutations. In addition, BCR signaling mutations are only detected in up to two-thirds of the CLL patients who progressed with BTKi treatment, suggesting mechanisms beyond genetic mutations. We previously showed that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells (Wang et al., Blood Cancer J. 2022), suggesting that epigenetic mechanisms are exploited by CLL B cells to support their survival. To gain insights into the epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome-wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on BTKi ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in a CLL cohort at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies showed that suppressing BCR signaling by BTKi leads to the downregulation of genes associated with mitochondrial translation, a key process of mitochondrial biogenesis. We also found decreased chromatin accessibility and H3K27ac deposition at the promoters of these MT genes. Interestingly, in patients who experienced disease progression on ibrutinib, there was a restoration of MT gene expression, chromatin accessibility, and H3K27ac levels at the MT gene promoters similar to baseline samples, suggesting an epigenetic-regulated MT both in BTKi treatment naïve and relapsed CLL patients. Importantly, MT inhibition led to a decreased survival of primary CLL B cells. These findings point toward an epigenetic mechanism utilized by BCR signaling to promote MT and leukemic cell survival. We then observed that the transcription factor NRF1 is enriched at the MT promoter in CLL B cells. CRISPR-mediated depletion of NRF1 led to reduced mitochondrial translation and cell survival in BCR signaling active malignant B cell lines and primary CLL B cells. These results suggest that NRF1 controls an epigenetic program linking BCR signaling to control MT, which in turn supports BCR signaling-dependent CLL B cell survival. Our findings demonstrated that BCR signaling utilizes epigenetic mechanisms that regulate mitochondrial metabolism to enhance cell survival in both treatment naïve and BTKi-relapsed CLL. These findings may provide new therapeutic directions for untreated and BTKi-relapsed CLL patients. Citation Format: Olivia M. Depies, Sutapa Sinha, Sameer Parikh, Neil Kay, Zhiquan Wang. An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia [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 5842.

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

Skewing Towards Effector Memory Cells in CLL Is Associated with IGHV -Mutation Status and IFNγ and IL-4

Memory T cells are a small, heterogenous population of T cells that remain following pathogen clearance and provide long-lasting protection. They are generally characterized as either central memory (T CM) or effector memory (T EM) based on differences in their surface and intracellular markers, anatomic location, effector function, and cytokine production. It is well known that patients with chronic lymphocytic leukemia (CLL) display differences in both T cell function and population compared to healthy individuals. Previous studies have shown that CLL patients skew towards the CD4 + memory compartment and among these, a skewing toward T EM is especially associated with unmutated IGHV and more progressive disease. Moreover, T EM cells appear to protect CLL cells, possibly via IFNγ and IL-4 mediated pathways. To understand this crosstalk between memory T cells and CLL B cells, we co-cultured FACS-purified T CM (CD3 +CD4 +CD19 -CCR7 +CD45RA -CD45RO +CD62L +) and T EM (CD3 +CD4 +CD19 -CCR7 -CD45RA -CD45RO +CD62L -) cells with CLL B cells and assessed shifts in the memory population as well as cytokines produced. Furthermore, as leukemic B-cell subpopulations differing in time since cell birth/division display distinct functional abilities to present antigen, we also investigated the effect of B-cell activation on this crosstalk. FACS-purified peripheral blood memory T cells and CLL B cells were obtained from treatment-naïve patient samples and co-cultured for 7 days to determine if the presence of CLL B cells, either resting (B rest) or pre-activated with CpG-ODN2006 and IL-15 (B act), would alter the T-cell population distribution. In the presence of either B rest or unmanipulated PBMC, overall, there were no significant changes in population of T EM or T CM. However, the distribution of T EM with B rest was bimodal and when grouped based on IGHV mutation status, we found significantly reduced counts of T EM in co-cultures with M-CLL (14.50% T EM, 11,019 cells) as compared to U-CLL (26.68% T EM, 20,648 cells) based on relative percentages and absolute cell counts ( P<0.001). Conversely, we saw an increase of T CM in M-CLL B rest co-cultures that correlated with decrease of T EM ( P<0.01). We then analyzed supernatants taken at the end of the 7-day co-culture period for IFNγ, IL-4, IL-17A, and IL-17F. We found elevated concentrations of IFNγ in co-cultures with both mutated-CLL and M- CLL, but significantly higher concentrations ( P<0.01) were found with U-CLL (312pg/ml) than M-CLL (173pg/ml). Similarly, IL-4 was elevated but more so (p<0.001) in U-CLL (88pg/ml) than M-CLL (44pg/ml). Of note, IFNγ and IL-4 have been implicated in progression of CLL with literature suggesting their role in prolonging CLL B cell survival. IL-17A and IL-17F were not elevated above baseline and no differences were found. Consistent with literature, the relative fraction of T EM in our patients negatively correlated with TTFT. We then repeated the experiments with pre-activated CLL B cells. When B act were co-cultured with memory T cells, we found significantly ( P<0.001) elevated absolute number of T EM and T CM. This indicated heightened proliferation of the memory population, with a greater degree of increase in the T EM population, resulting in a skewing toward T EM. Subsequent experiments found that co-culture with B act led to significantly elevated IL-4 and IFNγ levels. Interestingly, the effects mediated by B act, on both memory cell proliferation and cytokine production were markedly greater than with B rest. Moreover, unlike B rest, the effects of B act did not depend on IGHV mutation status but rather showed uniform increase in both T EM proliferation and IFNγ and IL-4 levels. Lastly, we analyzed the survival of CLL B cells at the end of the co-cultures. Overall, the addition of memory T cells significantly ( P<0.001) enhanced survival of CLL B cells and this effect was more prominent in B act (U-CLL: 51.1%, M-CLL: 40.3% survival) than B rest (U-CLL: 34.8%, M-CLL: 23.5% survival). Together, our in vitro results suggest crosstalk between memory T cells and CLL B cells in which CLL B cells, especially U-CLL, promote the expansion of T EM which is associated with shorter TTFT and more progressive disease. T EM, in turn, promote the survival of CLL B cells possibly through increased production of IFNγ and IL-4.

Transcriptomic Analyses Indicate That Pre-Leukemic B Cells from People with Monoclonal B Lymphocytosis Are Surprisingly Much Less Active Than Normal B Cells from People with Monoclonal B Lymphocytosis and from Healthy Donors

Introduction. Chronic lymphocytic leukemia (CLL) follows a stepwise developmental process, beginning in a specific group of normal B lymphocytes that undergo clonal expansion. These clonal expansions, known as monoclonal B lymphocytosis (MBL), are considered to be essential precursors of CLL based on immunologic, genetic, and epidemiologic studies. Since MBL represents the overabundance of a single CD5 + B cell, we tested how different the transcriptomes of MBL cells were from normal B cells (NBC) in people with MBL (NBC-MBL) and NBC in healthy donors (NBC-HD), expecting to identify activations pathways in the MBL cells that would explain clonal expansion. Methods. We collected and sorted specific cell populations from 13 patients with monoclonal B lymphocytosis (MBL): CD5 +CD19 +CD20 dimIgL-restricted MBL cells and CD5 -CD19 +CD20 + NBC. Additionally, we obtained CD5 -CD19 +CD20 + NBC from 13 healthy donors (NBC-HD). RNA was extracted and sequenced using SMART-Seq v4 Ultra Low Input and HiSeq platform. DESeq2 was used to analyze RNA-seq data. To identify genes with differential expression between these populations, the transcriptomes of MBL cells were compared to NBC from HD, employing a cutoff of P<0.05 and Log2fc ratio >± 1.5. We also used paired sample t-tests to compare 13 MBL cells to NBC from the same MBL patients. Significantly differentially expressed genes (DEG) resulting from these analyses were then subjected to Ingenuity Pathway Analysis (IPA). Results. When comparing the gene profiles of 13 MBL B cells to 13 NBC-HD cells, we identified 2695 DEG. Next, we compared gene profiles between MBL B cells and NBC-MBL cells obtained from 13 MBL patients, identifying 4821 genes. Using IPA, down-regulated B Cell Receptor Signaling, Cytokine Signaling, and Tumor Microenvironment Pathway were identified in both analyses. Upon overlapping the genes from these two analyses, we found that 803 genes passed the cutoff in both analyses. IPA analysis showed that 14 signaling pathways, including IL-33, IL-17, and HIF1a, were significantly downregulated in MBL cells. Moreover, the negative signaling pathway (Role of p14/p19ARF in Tumor Suppression) was upregulated in MBL cells. PDCD1 expression was significant higher in MBL (Log2Fc 6.8, P<0.0001) and 11 PDCD1 target genes were significantly regulated in MBL cells. IL-6 (Log2Fc -5.4, P<0.0001) expression was significantly lower in MBL and 47 IL-6 target genes were significantly regulated in MBL cells. Surprisingly, genes related to cell activation, proliferation and movement were largely downregulated in MBL cells. There were 2052 genes unique to the analysis MBL vs NBC-HD, IPA analysis showed several T-cell related signaling pathways e.g., T cell Exhaustion Signaling Pathway and T Lymphocyte Apoptosis were upregulated. In the 4018 genes unique in the analysis MBL vs NB-MBL. IPA results showed all the signaling pathways were downregulated in MBL. Discussion. Surprisingly, our transcriptomic analyses indicate that MBL cells are much less active than NBC from people with MBL and from HD. Specifically, we found that many cytokine signaling pathways and genes related with cell activation, proliferation and movement are downregulated in MBL cells. Also, PDCD1 is highly expressed in MBL cells, and PDCD1 target genes are also significantly regulated in MBL cells. This is consistent with the immunosuppressed features of MBL cells since interaction between PD-1 on B cells and its ligands in the microenvironment negatively influence the survival, proliferation, and immune evasion of CLL and MBL cells. Collectively, although functional studies are needed, these transcriptomic data strongly suggest that MBL cells are not in an activated state and are less active than even the NBC from the same person with MBL.

Melanoma and CLL co-occurrence and survival: role of KC history

BackgroundSurvival following melanoma and chronic lymphocytic leukemia (CLL) have both been individually associated with previous history of non-melanoma skin cancers (specifically keratinocyte carcinomas [KC]). Furthermore, melanoma and CLL have been reported to occur within the same patients. The survival experience of patients with both cancers is understudied, and the role of history of KC is unknown. Additional research is needed to tease apart the independent associations between KC and CLL survival, KC and melanoma survival, and the co-occurrence of all three cancers.MethodsA retrospective cohort study was conducted among patients who were diagnosed with melanoma and/or CLL at a comprehensive cancer center between 2008 and 2020. Multivariable Cox regression models were used to examine the association between history of KC and survival following melanoma and/or CLL with careful consideration of calendar year of diagnosis, treatment regimens and other risk factors. A nested case–control study comparing patients with both CLL and melanoma to those with only CLL or only melanoma was conducted to compare blood parameters across the three groups.ResultsA time-dependent association was observed between history of KC and favorable melanoma survival within 4 years following diagnosis and poorer survival post 7 years after melanoma diagnosis. History of KC was not significantly associated with survival following the diagnosis of CLL, after adjustment for clinical factors including historical/concurrent melanoma. Patients with co-occurring melanoma and CLL tended to be diagnosed with melanoma first and had elevated blood parameters including white blood cell and lymphocyte counts as compared with patients who were diagnosed with only melanoma.ConclusionsHistory of KC was an independent predictor of survival following melanoma but not of CLL. Additional studies are needed to determine if blood parameters obtained at the time of melanoma diagnosis could be used as a cost-effective way to identify those at high risk of asymptomatic CLL for the promotion of earlier CLL diagnosis.

CXC chemokine ligand 13 and galectin-9 plasma levels collaboratively provide prediction of disease activity and progression-free survival in chronic lymphocytic leukemia

The clinical outcome of lymphocytic leukemia (CLL) is quite heterogeneous. The purpose of this observational study was to investigate the clinical merit of measuring plasma galectin-9 and CXCL-13 concentrations as predictors of CLL activity, prognosis, and early indicators of therapeutic response. These biomarkers were compared with other prognostic indicators, progression-free survival (PFS), time to first treatment (TTT), and overall survival (OS) over a follow-up period (4 years). First, plasma galectin-9 and CXCL-13 concentrations were analyzed in CLL patients at the time of diagnosis as well as healthy controls. Compared to controls, CLL patients had significantly higher serum levels of CXCL-13 and galectin-9. Second, we observed that CLL patients with high soluble CXCL-13 and galectin-9 levels had advanced clinical stages, poor prognosis, 17p del, short PFS, short TTT, and therapy resistance. The levels of CXCL-13, β2-microglobulin, LDH, CD38%, and high grade of Rai-stage were all strongly correlated with the galectin-9 levels. Soluble CXCL-13 and galectin-9 had very good specificity and sensitivity in detecting CLL disease progression and high-risk patients with the superiority of galectin-9 over CXCL-13. Although the two biomarkers were equal in prediction of TTT and treatment response, the soluble CXCL13 was superior in prediction of OS. High CXCL-13 and galectin-9 plasma levels upon CLL diagnosis are associated with disease activity, progression, advanced clinical stages, short periods of PFS, short TTT, and unfavorable treatment response.

High-throughput Proteomics Identifies THEMIS2 as Independent Biomarker of Treatment-free Survival in Untreated CLL.

It remains challenging in chronic lymphocytic leukemia (CLL) to distinguish between patients with favorable and unfavorable time-to-first treatment (TTFT). Additionally, the downstream protein correlates of well-known molecular features of CLL are not always clear. To address this, we selected 40 CLL patients with TTFT ≤24 months and compared their B cell intracellular protein expression with 40 age- and sex-matched CLL patients with TTFT >24 months using mass spectrometry. In total, 3268 proteins were quantified in the cohort. Immunoglobulin heavy-chain variable (IGHV) mutational status and trisomy 12 were most impactful on the CLL proteome. Comparing cases to controls, 5 proteins were significantly upregulated, whereas 3 proteins were significantly downregulated. Of these, only THEMIS2, a signaling protein acting downstream of the B cell receptor, was significantly associated with TTFT, independently of IGHV and TP53 mutational status (hazard ratio, 2.49 [95% confidence interval, 1.62-3.84]; P < 0.001). This association was validated on the mRNA and protein level by quantitative polymerase chain reaction and ELISA, respectively. Analysis of 2 independently generated RNA sequencing and mass spectrometry datasets confirmed the association between THEMIS2 expression and clinical outcome. In conclusion, we present a comprehensive characterization of the proteome of untreated CLL and identify THEMIS2 expression as a putative biomarker of TTFT.

Expression and Activity of the NF-κB Subunits in Chronic Lymphocytic Leukaemia: A Role for RelB and Non-Canonical Signalling.

Canonical NF-κB signalling by p65 (RelA) confers chemo-resistance and poor survival in chronic lymphocytic leukaemia (CLL). The role of non-canonical NF-κB signalling (leading to RelB and p52 subunit activation) in CLL is less understood, but given its importance in other B-cell tumour types, we theorised that RelB and p52 may also contribute to the pathology of CLL. DNA binding activity of all five NF-kB subunits, p65, p50, RelB, p52, and c-Rel, was quantified using ELISA and correlated to ex vivo chemoresistance, CD40L-stimulated signalling (to mimic the lymph node microenvironment), and clinical data. Importantly, we show for the first time that high basal levels of RelB DNA binding correlate with nuclear RelB protein expression and are associated with del(11q), ATM dysfunction, unmutated IGHV genes, and shorter survival. High levels of nuclear p65 are prevalent in del(17p) cases (including treatment-naïve patients) and also correlate with the outcome. CD40L-stimulation resulted in rapid RelB activation, phosphorylation and processing of p100, and subsequent CLL cell proliferation. These data highlight a role for RelB in driving CLL cell tumour growth in a subset of patients and therefore strategies designed to inhibit non-canonical NF-κB signalling represent a novel approach that will have therapeutic benefit in CLL.

In Vitro 3D Spheroid Culture System Displays Sustained T Cell-dependent CLL Proliferation and Survival.

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental cells and signals. The lymph node (LN) is the critical site of in vivo CLL proliferation and development of resistance to both chemotherapy and targeted agents. We present a new model that incorporates key aspects of the CLL LN, which enables investigation of CLL cells in the context of a protective niche. We describe a three-dimensional (3D) in vitro culture system using ultra-low attachment plates to create spheroids of CLL cells derived from peripheral blood. Starting from CLL:T cell ratios as observed in LN samples, CLL activation was induced by either direct stimulation and/or indirectly via T cells. Compared with two-dimensional cultures, 3D cultures promoted CLL proliferation in a T cell-dependent manner, and enabled expansion for up to 7 weeks, including the formation of follicle-like structures after several weeks of culture. This model enables high-throughput drug screening, of which we describe response to Btk inhibition, venetoclax resistance, and T cell-mediated cytotoxicity as examples. In summary, we present the first LN-mimicking in vitro 3D culture for primary CLL, which enables readouts such as real-time drug screens, kinetic growth assays, and spatial localization. This is the first in vitro CLL system that allows testing of response and resistance to venetoclax and Bruton's tyrosine kinase inhibitors in the context of the tumor microenvironment, thereby opening up new possibilities for clinically useful applications.

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.

Targeting conditioned media dependencies and FLT-3 in chronic lymphocytic leukemia

AbstractThe importance of the stromal microenvironment in chronic lymphocytic leukemia (CLL) pathogenesis and drug resistance is well established. Despite recent advances in CLL therapy, identifying novel ways to disrupt interactions between CLL and its microenvironment may identify new combination partners for the drugs currently in use. To understand the role of microenvironmental factors on primary CLL cells, we took advantage of an observation that conditioned media (CM) collected from stroma was protective of CLL cells from spontaneous cell death ex vivo. The cytokine in the CM-dependent cells that most supports CLL survival in short-term ex vivo culture was CCL2. Pretreatment of CLL cells with anti-CCL2 antibody enhanced venetoclax-mediated killing. Surprisingly, we found a group of CLL samples (9/23 cases) that are less likely to undergo cell death in the absence of CM support. Functional studies revealed that CM-independent (CMI) CLL cells are less sensitive to apoptosis than conventional stroma-dependent CLL. In addition, a majority of the CMI CLL samples (80%) harbored unmutated immunoglobulin heavy-chain variable (IGHV) region. Bulk-RNA sequence analysis revealed upregulation of the focal adhesion and RAS signaling pathways in this group, along with expression of fms-like tyrosine kinase 3 (FLT3) and CD135. Treatment with FLT3 inhibitors caused a significant reduction in cell viability among CMI samples. In summary, we were able to discriminate and target 2 biologically distinct subgroups of CLL based on CM dependence with distinct microenvironmental vulnerabilities.

Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

It is well established that inflammation and platelets promote multiple processes of cancer malignancy. Recently, platelets have received attention for their role in carcinogenesis through the production of microvesicles or platelet-derived microparticles (PMPs), which transfer their biological content to cancer cells. We have previously characterized a new subpopulation of these microparticles (termed mito-microparticles), which package functional mitochondria. The potential of mitochondria transfer to cancer cells is particularly impactful as many aspects of mitochondrial biology (i.e., cell growth, apoptosis inhibition, and drug resistance) coincide with cancer hallmarks and disease progression. These metabolic aspects are particularly notable in chronic lymphocytic leukemia (CLL), which is characterized by a relentless accumulation of proliferating, immunologically dysfunctional, mature B-lymphocytes that fail to undergo apoptosis. The present study aimed to investigate the role of PMPs on CLL metabolic plasticity leading to cancer cell phenotypic changes. CLL cell lines were co-incubated with different concentrations of human PMPs, and their impact on cell proliferation, mitochondrial DNA copy number, OCR level, ATP production, and ROS content was evaluated. Essential genes involved in metabolic-reprogramming were identified using the bioinformatics tools, examined between patients with early and advanced CLL stages, and then validated in PMP-recipient CLLs. Finally, the impact of the induced metabolic reprogramming on CLLs' growth, survival, mobility, and invasiveness was tested against anti-cancer drugs Cytarabine, Venetoclax, and Plumbagin. The data demonstrated the potency of PMPs in inducing tumoral growth and invasiveness in CLLs through mitochondrial internalization and OXPHOS stimulation which was in line with metabolic shift reported in CLL patients from early to advanced stages. This metabolic rewiring also improved CLL cells' resistance to Cytarabine, Venetoclax, and Plumbagin chemo drugs. Altogether, these findings depict a new platelet-mediated pathway of cancer pathogenesis. We also highlight the impact of PMPs in CLL metabolic reprogramming and disease progression.

HDAC1 regulates the chromatin landscape to control transcriptional dependencies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a quiescent B-cell malignancy that depends on transcriptional dysregulation for survival. The histone deacetylases are transcriptional regulators whose role within the regulatory chromatin and consequence on the CLL transcriptome is poorly characterized. Here, we profiled and integrated the genome-wide occupancy of HDAC1, BRD4, H3K27Ac, and H3K9Ac signals with chromatin accessibility, Pol2 occupancy, and target expression signatures in CLL cells. We identified that when HDAC1 was recruited within super-enhancers (SEs) marked by acetylated H3K27 and BRD4, it functioned as a transcriptional activator that drove the de novo expression of select genes to facilitate survival and progression in CLL. Targeting HDACs reduced BRD4 and Pol2 engagement to downregulate the transcript and proteins levels of specific oncogenic driver genes in CLL such as BLK, a key mediator of the B-cell receptor pathway, core transcription factors such as PAX5 and IKZF3, and the antiapoptotic gene, BCL2. Concurrently, HDAC1, when recruited in the absence of SEs, repressed target gene expression. HDAC inhibition reversed silencing of a defined set of protein-coding and noncoding RNA genes. We focused on a specific set of microRNA genes and showed that their upregulation was inversely correlated with the expression of CLL-specific survival, transcription factor, and signaling genes. Our findings identify that the transcriptional activator and repressor functions of HDACs cooperate within the same tumor to establish the transcriptional dependencies essential for survival in CLL.

Abstract 4725: The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia

Abstract Introduction: The introduction of Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) to target B cell receptor (BCR) pathways in chronic lymphocytic leukemia (CLL) has significantly improved clinical responses. However, the effectiveness of BTKi is limited by the development of adaptive drug resistance. Therefore, there is an urgent need to identify critical biologic features that determine BTKi efficacy in order to develop more effective therapies. Epigenetic aberrations play an important role in tumor initiation, progression, and drug resistance. However, it is currently unknown if BCR signaling is also dependent on epigenetic mechanisms to support oncogenic gene expression and survival in CLL B cells. Methods and Results: To gain new insights into the role of epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&amp;Tag) of leukemic cells from 4 CLL patients on ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in other cohorts at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies show that suppressing BCR signaling by BTKi treatment leads to an alteration of a pro-survival epigenetic signature (defined by histone modifications and chromatin accessibility landscapes) in CLL and that the genes regulated by these chromatin changes are enriched in pathways associated with malignant B cell survival (i.e., BCR signaling and apoptosis pathways). Importantly, disruption of this pro-survival epigenetic signature is necessary for effective BTKi treatment. We further find that the transcription factor (TF) Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) couples the BCR signaling to control this pro-survival epigenetic program that supports CLL B cell survival. In specific, we show that nuclear depletion of NFATc1 is required for BTKi induced epigenetic alteration and effective BTKi treatment. Depletion of NFATc1 disrupts the pro-survival epigenetic signature and inhibits CLL B cells survival, which improves the efficacy of BTKi treatment in CLL. Conclusion: Here using leukemic cells from CLL patients with BTKi treatment, we demonstrate that epigenetic regulators are exploited by the oncogenic signaling pathway to support malignant cell survival. Specifically, NFATc1 is utilized by the BCR signaling pathway to control an epigenetic signature that supports CLL cell survival. This BCR regulated epigenetic machinery can be targeted to maintain and enhance BTKi treatment efficacy in CLL. Citation Format: Zhiquan Wang, Huihuang Yan, Justin C. Boysen, Esteban Braggio, Sameer A. Parikh, Neil E. Kay. The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4725.

Abstract 2516: Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL

Abstract Microenvironments such as lymph nodes and bone marrow allow chronic lymphocytic leukemia (CLL) cells to survive after drug treatments. There are limited methods to study the to study the contribution of the microenvironment. We have adapted a solid tumour microenvironment cell culture system that provides elements of the CLL microenvironment called Analysis of CLL Cellular Environment and Response (ACCER). We optimized the cell number for patient’s primary CLL cells and HS-5 human bone marrow stromal cell line that will give sufficient cell number and viability with the ACCER. We then determined the amount of collagen type 1 to give the best extracellular matrix to seed CLL cells to the membrane. Finally, we determined that ACCER provide CLL cell protection against cell death following treatment with fludarabine and ibrutinib compared to co-culture conditions. This describes novel microenvironment model to investigate factors that promote drug resistance and cell survival in CLL. In the future, this will further validate new treatment strategies to overcome microenvironmental CLL cell survival signals. Citation Format: Spencer Gibson, Tricia Choquette, Elizabeth S. Henson, Xioyan Yang, James B. Johnston. Analysis of CLL Celllular Environment and Response (ACCER) is a novel method to understand the microenvironment in CLL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2516.

PYK2 is overexpressed in chronic lymphocytic leukaemia: Apotential new therapeutic target.

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.

Time to second treatment can be used to predict overall survival in chronic lymphocytic leukemia: identifying risk factors to help guide treatment selection

Targeted therapies have largely replaced chemoimmunotherapy (CIT) in first-line treatment of chronic lymphocytic leukemia (CLL). We aimed to develop a prognostic model to determine who would benefit from first-line CIT vs target therapy. In follicular lymphoma, time from diagnosis to second treatment (TT2T) correlates better with overall survival (OS) than time from diagnosis to first treatment (TT1T). We hypothesized that TT2T is a potential surrogate for OS in CLL. In a model-building cohort (n = 298), we evaluated potential predictors for TT2T and derived a risk score, which we validated in an external cohort (n = 1141). Our data demonstrated that TT2T and OS were more strongly correlated than TT1T and OS. Our risk score model consisted of three predictors (unmutated IGHV, β2-microglobulin >297 nmol/L, and Rai stage I–IV), and was prognostic for TT2T and OS. TT2T is a promising surrogate for OS in CLL, but further validation is needed to establish this association.

CLECL1+ CLL Cells: A Key Subset That Drives CLL Survival and Proliferation and Shapes a Th2-Biased Tumor Microenvironment

Chronic lymphocytic leukemia (CLL) cells depend on elements in the tissue microenvironment (TME) that produce factors such as IL-4 to promote leukemic B cell survival and growth. CLECL1, a C-type lectin-like protein, is a T cell co-stimulatory molecule originally identified on dendritic cells, but also expressed on normal B and myeloid cells. Interactions of CLECL1 with naïve T cells (Tn) lead to Th2 cell development and production of IL-4. While IL-4 can have pro- and anti- tumor activities, a role for CLECL1 in tumorigenesis has not been tested. Here we studied the role of CLECL1 in CLL. Flow cytometry analyses of CD19+CD5+ cells from 7 normal people and 77 patients with CLL revealed significantly higher CLECL1 levels on CLL cells (CLL vs Normal = 10.1% vs 3.9%, P = 0.01). CLECL1 was found most in the fraction of leukemic clones enriched in recently divided cells (PF; CXCR4DimCD5Bright) and least in the resting fraction (RF; CXCR4BrightCD5Dim) (PF vs RF = 20% vs 0.4%, P < 0.001). Importantly, high levels of CLECL1 in the PF correlated with shorter time to first treatment (P = 0.01). Notably, effective treatment with ibrutinib significantly reduced CLECL1+ cells (pre vs post treatment: 9.1% vs 1.8%, P = 0.01), and these levels increased when patients developed ibrutinib resistance (sensitive vs resistance = 1.8% vs 7.5%, P < 0.05). Flow cytometry analyses also indicated that, compared to CLECL1- cells, CLECL1+ CLL cells expressed higher levels of other cell interaction molecules such as CD40 and CD80, as well as activation markers, CD23, CD27, CD69, CD71 and CD72. Consistent with this, CLECL1+ PF cells were mainly in the G2/M phase of the cell cycle and express AID. In contrast, CLECL1- PF cells were AID- and chiefly in G1/S phases or undergoing apoptosis. Single cell RNAseq analyses of CLL PF versus RF cells corroborated that the expression of CLECL1 is [1] restricted to the PF, [2] associated with genes expressed in G2/M, and [3] correlated with IL-4-related gene expression. Together, these data suggest CLECL1+ CLL cells are activated cells generated from CLECL1- cells. Indeed, stimulation of CLECL1- cells in vitro revealed that anti-IgM+IL-4 converted CLECL1- to CLECL1+ cells. In CLL patient lymph nodes, CLECL1+ cells expressed Ki67 and co-localized with T cells. Thus, we questioned the abilities of CLECL1+ and CLECL1- cells to interact with T cells and to affect T cell growth and cytokine production. Co-culture of unseparated autologous T cells with CLECL1+, but not CLECL1- CLL cells significantly enhanced proliferation of T cells stimulated through T-cell receptors (CLECL1+ vs CLECL1-: 12.9% vs 2.8%, P = 0.02) and induced increased production of IL-4 (CLECL1+ vs CLECL1-: 15% vs 7%, P < 0.05). Contrary to published results that recombinant CLECL1 (rCLECL1) only binds to healthy Tn, we identified direct binding of rCLECL1 to both Tn and T memory (Tmem) cells in CLL patients. Moreover, when Tn and Tmem cells were incubated with rCLECL1, both T cell subsets responded with enhanced proliferation and IL-4 production. Subsequently, we tried to recapitulate the above in vitro findings in vivo, using patient derived xenografts. Injection of NSG mice with autologous CD3+ T cells plus CLECL1+ or CLECL1- PF cells led to significantly higher numbers of CLL B and T cells in the spleens of recipient mice (CLECL1+ vs CLECL1- for B cells: 508,800 vs 37,000; P = 0.03; for T cells: 273,700 vs 47,000; P = 0.03). Additionally, Th2 cells outgrew Th1 T cells (CLECL1+ vs CLECL1- for Th2 cells: 60.1% vs 12%; P < 0.05; for Th1 cells: 20.1% vs 1.8%; N.S). The same results were seen when we injected CLECL1+ or CLECL1- CLL PF cells plus autologous Tn or Tmem cells into NSG mice, as only CLECL1+ PF cells stimulated Tn or Tmem cells to expand and produce IL-4. In turn, these CLECL1 stimulated T cells facilitated the growth of CLL cells in recipient spleen and bone marrow. Overall, these ex vivo, in vitro, and in vivo results indicate that CLL cells that express CLECL1, specifically those in the recently-divided fraction, engage both Tn and Tmem cells to promote T cell growth and IL-4 production. Consequently, the CLECL1-shaped Th2 TME facilitates the survival and proliferation of tumor cells. Therefore, despite the relatively small numbers, CLECL1+ cells in the PF of CLL clones are potential key targets for therapy. Eliminating CLECL1+ CLL cells or blocking their promotion of IL-4 production could improve treatment, especially for those patients with CLL resistant to ibrutinib.

CLL-177 Elevated Serum ELABELA and SERPINA3 as Novel Biomarkers to Predict Poor Prognosis in Egyptian Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL) is the most common kind of adult leukemia worldwide. Increased expressions of serpin peptidase inhibitor clade A member 3 (SERPINA3) and apelin receptor early endogenous ligand (ELABELA) have been observed in various malignancies, however, there is little information on their clinical importance in CLL. The purpose of this study was to determine serum levels of SERPINA3 and ELABELA in CLL patients. Prospective study performed from April 2020 to March 2022. This study was conducted at Oncology Center, Mansoura University, Mansoura, Egypt. The study included 67 newly diagnosed CLL patients and 66 healthy individuals as a control group. The CLL patients indicated for therapy received Fludarabine, Cyclophosphamide, and Rituximab (FCR) for 17 p deletion (del.17p) negative eligible patients, Chlorambucil for del.17p negative non-eligible patients, and Ibrutinib or high-dose Methylprednisolone (HDMP) for del.17p positive patients. The serum levels of SERPINA3 and ELABELA were determined using an enzyme-linked immunosorbent assay. To detect the impact of serum levels of SERPINA3 and ELABELA on prognosis and survival of CLL patients. Our study found that serum levels of SERPINA3 and ELABELA were significantly higher in CLL patients than in the control group with p<0.001 and p=0.001, respectively. There was a significant increase in serum levels of SERPINA3 and ELABELA in Rai IV and Binet C stages with p<0.001 for each. Both ELABELA and SERPINA3 levels were significantly higher in CLL patients with positive del.17p compared to negative patients with p=0.04 and p=0.008, respectively. Both increased levels of SERPINA3 and ELABELA were associated with decreased response rates and shortened progression-free survival (PFS) and overall survival (OS). Only ELABELA was an independent predictor of shorter PFS in univariate analysis. SERPINA3 and positive del.17p were significant predictors of shorter OS in univariate analysis. In multivariate analysis, only SERPINA3 was an independent risk factor for shorter OS. SERPINA3 and ELABELA are poor prognostic biomarkers in CLL patients associated with dismal outcomes. SERPINA3 and ELABELA may be a promising therapeutic target for CLL treatment in the future.