Disease Progression In Chronic Lymphocytic Leukemia Research Articles

Lessons learned from the Eµ-TCL1 mouse model of CLL

The Eµ-TCL1 mouse model has been used for over 20 years to study the pathobiology of chronic lymphocytic leukemia (CLL) and for preclinical testing of novel therapies. A CLL-like disease develops with increasing age in these mice due to a B cell specific overexpression of human TCL1. The reliability of this model to mirror human CLL is controversially discussed, as none of the known driver mutations identified in patients are found in Eµ-TCL1 mice. It has to be acknowledged that this mouse model was key to develop targeted therapies that aim at inhibiting the constitutive B cell receptor (BCR) signaling, a main driver of CLL. Inhibitors of BCR signaling became standard-of-care for a large proportion of patients with CLL as they are highly effective. The Eµ-TCL1 model further advanced our understanding of CLL biology owed to studies that crossed this mouse line with various transgenic mouse models and demonstrated the relevance of CLL-cell intrinsic and -extrinsic drivers of disease. These studies were instrumental in showing the relevance of the tumor microenvironment in the lymphoid tissues for disease progression and immune escape in CLL. It became clear that CLL cells shape and rely on stromal and immune cells, and that immune suppressive mechanisms and T cell exhaustion contribute to CLL progression. Based on this knowledge, new immunotherapy strategies were clinically tested for CLL, but so far with disappointing results. As some of these therapies were effective in the Eµ-TCL1 mouse model, the question arose concerning the translatability of preclinical studies in these mice. The aim of this review is to summarize lessons we have learnt over the last decades by studying CLL-like disease in the Eµ-TCL1 mouse model. The article focuses on pitfalls and limitations of the model, as well as the gained knowledge and potential of using this model for the development of novel treatment strategies to achieve the goal of curing patients with CLL.

Mouse models of CLL: In vivo modeling of disease initiation, progression, and transformation to Richter transformation

Chronic lymphocytic leukemia (CLL) is a highly complex disease characterized by the proliferation of CD5+ B cells in lymphoid tissues. Current modern treatments have brought significant clinical benefits to CLL patients. However, there are still unmet needs. Patients relapse on Bruton's tyrosine kinase inhibitors and BCL2 inhibitors and often develop more aggressive diseases including Richter transformation (RT), an incurable complication of up to ∼10% patients. This evidence underscores the need for improved immunotherapies, combination treatment strategies, and predictive biomarkers. A mouse model that can recapitulate human CLL disease and certain components of the tumor immune microenvironment represents a promising preclinical tool for such purposes. In this review, we provide an overview of CRISPR-engineered and xenograft mouse models utilizing either cell lines, or primary CLL cells suitable for studies of key events driving the disease onset, progression and transformation of CLL. We also review how CRISPR/Cas9 established mouse models carrying loss-of-function lesions allow one to study key mutations driving disease progression. Finally, we discuss how next generation humanized mice might improve to generation of faithful xenograft mouse models of human CLL.

Abstract 297: Role of purine metabolism in CLL cell pathobiology and CLL disease progression

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

Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Progressive Gut Microbiome Dysbiosis Is Associated with Chronic Lymphocytic Leukemia Pathogenesis and Development in the Eµ-TCL1 Mouse Model

Introduction: The gut microbiome is home to a community of microorganisms such as bacteria, fungi, viruses, archaea, and protozoa. Maintaining a balance between “favorable” and “unfavorable” bacteria within the gut microbiome is essential for homeostatic physiological processes including proper immune system development, regulation of metabolic pathways, protection against pathogens, and gut epithelium integrity. Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by the accumulation of CD5 + B-cells in the blood, bone marrow, and secondary lymphoid tissues. The pathogenesis of CLL is related to the activation of critical survival pathways such as B-cell and toll-like receptor signaling pathways, where microbial antigens are known to be key stimulators driving CLL B-cell proliferation. In this study, we investigated how the gut microbiota composition contributes and correlates to CLL disease progression. Methods: Using a well-established murine model representative of CLL development in humans, we profiled the gut microbiota of transgenic Eµ-TCL1 mice and age-matched wild-type B6 (WT B6) mice over the course of 12 months (n = 7-10/group). Fecal pellets were collected aseptically from study mice at four time points (4, 7, 10, and 12 months) for DNA extraction using the QIAamp® PowerFecal® Pro DNA Kit. To characterize the gut microflora, we utilized 16S rRNA sequencing using the Illumina 16S metagenomics protocol. Bacterial taxonomy was assigned and analyzed using the QIIME2™ platform. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analysis was employed to identify differentially expressed bacterial taxa between cohorts. Beta diversity indices were used to evaluate intersample differences in the fecal microbiota. Mice were monitored for peripheral expansion of leukemia cells (%CD45 +/CD19 +/CD5 + cells) via flow cytometry. Next, we evaluated the microbiome in an aggressive model of CLL. Utilizing the Eµ-TCL1 adoptive transfer murine model, similar methods were adopted to profile the gut microbiome over the course of 7 weeks. Lastly, to evaluate the contribution of the gut microbiota to CLL development, we employed an antibiotic-mediated gut microflora ablation to the adoptive transfer Eµ-TCL1 model, with leukemia expansion monitored post-engraftment on a weekly basis. Results: A unique microbiome signature was identified in both the indolent and aggressive model of CLL. Evaluation of beta diversity, using Bray-Curtis distance metrics, confirmed distinct differences in the gut microbial communities between Eµ-TCL1 and WT B6 cohorts. At the phylum level, the gut microbiota of the Eµ-TCL1 and WT B6 cohorts were similarly dominated by Firmicutes and Bacteroidota. However, Proteobacteria Deferribacterota and Desulfobacterota were more abundant in the Eµ-TCL1 mice over 12 months as compared to age-matched WT B6. Abundance of Actinobacteriota increased specifically at 12 months in the Eµ-TCL1 mice. Additionally, LEfSe analysis showed enrichment of certain phyla in the Eµ-TCL1 mice at 4 months, 10 months, and 12 months. Presence of these bacteria paralleled with expansion of disease. In the aggressive model of CLL, beta diversity confirmed distinct differences in gut microbial communities as CLL disease progressed. Moreover, certain phyla were enriched in leukemic mice at 7 weeks post-engraftment including Proteobacteria and Actinobacteriota. Lastly, in the antibiotic-mediated gut flora ablation model, a delay of disease onset was reflected in mice with ablated gut microbiomes compared to mice with intact gut microbiomes, resulting in prolonged overall survival. Conclusion: We provide novel results associating the gut microbiome with the initiation and progression of CLL disease. In both the indolent and aggressive CLL disease models, we showed that there is a distinct gut microbiome as evident by beta diversity. A dysbiotic signature represented by increasing Proteobacteria abundance was similarly identified in both models. Proteobacteria are known to be evident in diseases characterized by inflammation. These findings support future studies to determine a potential causal mechanism between gut microbiota changes and CLL pathogenesis.

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 >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<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.

Prediction of Time-to-First Treatment (TTFT) in Favorable-Risk Chronic Lymphocytic Leukemia (CLL) Patients Using a Novel Stratification through Transcriptome Risk Scoring

Background: CLL is the most prevalent adult leukemia with a highly heterogenous genetic landscape leading to extremely varied clinical outcomes. Previously, CLL patients with immunoglobulin heavy chain variable region ( IGHV) mutated status and presence of deletion 13q14 [del(13q)] were identified as favorable-risk with most patients remaining on lifetime observation. However, other intrinsic factors contributing to CLL disease progression and leading to treatment initiation remain unknown. Our goal was to identify patients at high-risk of progression with added predictive value (APV) beyond IGHV-status and presence of del(13q). Methods: Flow cytometry was used to sort CD19+CD5+ malignant cells in peripheral blood from CLL patients at Huntsman Cancer Institute at University of Utah and triple sequencing generated (tumor whole transcriptome and paired tumor-normal whole exome data). We used SPECTRA, a novel dimension reduction transcriptome approach, to derive a set of independent, intrinsic, quantitative transcriptome tumor variables from 249 CLL patients with quality-controlled transcriptome sequence data. From this set, 182 treatment-naïve (TN) patients with quality-controlled clinical variables were used as a discovery cohort set to derive a SPECTRA-based risk score for TTFT using multivariable Cox proportional hazards modeling. Event time was date of patient specimen acquisition to initiation of frontline therapy with patients censored at last follow-up or death. Gaussian mixture modeling (GMM) was performed to identify evidence for distinct risk groups within the quantitative risk score. GMM-risk groups, and further stratifications by IGHV mutational and del(13q) status, were used in hazard ratios (HR) and to illustrate differences in Kaplan-Meier time-to-event curves. An independent publicly available dataset of 548 TN CLL patients with clinical and transcriptome data for CD19+ enriched cells was used for replication. Results: SPECTRA produced 19 quantitative spectra variables to describe most of the expression variation across the 249 CLL patients. In multivariable Cox modeling for TTFT, 7 spectra were significant and retained in a final model, which GMM separated into a bi-modal distribution and defined high (N=83) and low-risk (N=99) groups (HR=5.7, 95% confidence interval [CI] 3.4-9.7). In a post-hoc model including IGHV mutational and del(13q) status, the spectra-risk score showed significant APV beyond both factors (p<0.001 for both). The discovery study timeframe was 6.3 years (y) and included 109 IGHV-mutated and 75 del(13q) patients. In the IGHV-mutated patients, median TTFT (mTTFT) was significantly shorter for spectra-high risk patients (3.1y) vs. spectra-low risk patients (median not reached [NR]; p<0.001). Similarly, in the del(13q) patients, mTTFT was significantly shorter in high-risk patients (3.3y) compared to median NR for spectra-low (p<0.001) ( Figure). Importantly, stratification of patients into low- and high-risk SPECTRA scores provided significant additional information for prediction of TTFT than IGHV mutational and del(13q) status ( Figure). For validation, we applied the same spectra-risk score to the independent data set containing 301 IGHV-mutated and 278 del(13q) CLL patients. In the replication set, the mTTFT in the IGHV-mutated patients who were spectra-high was 3.3y vs. 11.2y in spectra-low (p<0.001). Similarly, in the replication set of del(13q) patients, spectra-high-risk patients had a mTTFT of 2.7y vs. 10.7y for spectra-low (p<0.001). Conclusion: A transcriptome risk score utilizing novel SPECTRA variables identified a substantial subset of CLL patients with IGHV-mutated status and presence of del(13q) who have uncharacteristically short TTFT, but who would be classified as favorable risk under current best practice stratification. Future directions include validating our results with external patient cohorts from Mayo Clinic and investigating the genetic and transcriptomic profile of SPECTRA low- and high-risk cohorts to determine targetable pathways for treatment.

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.

Dysregulation of PRMT5 in Chronic Lymphocytic Leukemia Supports Transformation to Diffuse Large B-Cell Lymphoma

Background: Richter's transformation (RT) is the progression of chronic lymphocytic leukemia (CLL) to a high-grade lymphoma, most commonly resembling a diffuse large B-cell-like lymphoma, that has a bleak prognosis of 6-12 months. Many of the recurrent somatic alterations identified in RT are also associated with the CLL-phase of disease, suggesting additional unidentified genetic, epigenetic, or molecular events underlie transformation. Aberrant PRMT5 expression is known to promote and maintain oncogenic signaling in non-Hodgkin's lymphomas but its role in CLL and RT remains unclear. Methods: Tissue microarrays and western blots were used to evaluate PRMT5 expression in CLL/SLL and RT lymph nodes (LN). Differentially expressed genes between patient-matched RT LNs and CLL PBMCs were identified with scRNA-seq & scV(D)J-seq. ScRNA-seq was utilized to identify transcriptional changes between Eμ-PRMT5/TCL1 & Eμ-TCL1 mice. Tumor allografts were generated by administering 5 x 10^6 CD19+CD5+ spleen cells from Eμ-PRMT5/TCL1 mice intravenously to C57/BL6J mice. One week post-engraftment mice were enrolled in treatment groups (PRT382 10mg/kg, n=10; vehicle, n=7) with all treatments administered for 4 contiguous days per week by oral gavage. Results: Tissue microarray analysis on 70 CLL and 15 RT LN cases showed robust PRMT5 staining in RT LNs and minimal staining in CLL tissues. Immunoblot analysis revealed that CLL cells from patients that eventually undergo transformation displayed significant increase in PRMT5 expression months prior to transformation, whereas CLL patients maintained variable to minimal PRMT5 expression. ScRNA-seq & scV(D)J-seq on RT LN biopsies and CLL PBMCs from two patients revealed that PRMT5 expression was restricted to distinct nodal B cell subpopulations co-expressing MYC, BIRC5, CD83, and CD69. One PRMT5+ cluster displayed overlapping expression of proliferative markers (Mki67, TOP2A, PCNA, CALM2/3, and HMGB1/2) with enriched EIF4 signaling. Anti-apoptotic and immune modulating genes (BCL21A, IL4I1, TCL1A, and CCL3/4) were enriched in the second PRMT5+ nodal cluster. Interestingly, transcriptional diversity was enhanced in clonally related RT B cell populations with an increased proportion of cells expressing PRMT5 when compared to the CLL-phase of disease. To further evaluate the role of PRMT5 in CLL disease progression, we conducted histopathologic analysis on the spleen and lymph nodes of 6 month old animals from Eµ-PRMT5/TCL1 & Eµ-TCL1 mouse models, revealing a RT-like histology with loss of germinal centers and effacement of normal architecture exclusively in Eµ-PRMT5/TCL1 animals. Transcriptional changes underlying phenotypic differences between Eµ-PRMT5/TCL1 & Eµ-TCL1 mice were also assessed by scRNA-seq, where splenic B cells of the Eµ-PRMT5/TCL1 exhibited increased expression of oncogenic and immune regulating genes (Myc, Mki67, Egr1, Cxcr5, Ccr7, Il-10, Ctla4, and Pd-L1). Interestingly, the dominant B-cell clusters enriched in Eµ-PRMT5/TCL1 spleen and LNs showed significant transcriptional overlap with human RT PRMT5 expressing LN clusters (e.g., Myc, Cd83, and Cd69). With the observation that PRMT5 may play a significant role in CLL-to-RT evolution, we then aimed to evaluate the use of targeted PRMT5 inhibition against murine RT-like tumors in vivo. Using the selective SAM-competitive PRMT5 inhibitor, PRT382, in Eµ-PRMT5/TCL1 allografts, we observed PRT382 treatment delayed leukemic expansion, increased median survival (p<0.0001), and reduced spleen mass compared to vehicle treated mice. Conclusion: We show that PRMT5 is upregulated in the lymph nodes of CLL patients prior to and after transformation, suggesting a role for PRMT5 in CLL transformation to RT. Likewise, the Eμ-PRMT5/TCL1 mouse model develops a RT-like histology and shows significant overlap in the transcriptional profile to human RT tumors. And further, inhibition of PRMT5 with PRT382 provides significant improvement in median overall survival and slows disease progression in Eμ-PRMT5/TCL1 adoptive transfer models. Our preclinical data identifies PRMT5 as a high-risk marker in CLL and provides rationale for the clinical evaluation of PRMT5 inhibition in the treatment of high-risk CLL/RT.

Anti-CD70 Monoclonal Antibodies Show Efficacy in Preclinical Models for Chronic Lymphoblastic Leukemia

Although therapy for chronic lymphocytic leukemia (CLL) has improved significantly with combination therapies that include ibrutinib and/or anti-B cell mAbs, resistance can develop, and patients can discontinue therapy for various reasons. Therefore, effective, alternative therapies are needed. CD70, a TNF family member, and its receptor, CD27, are mainly expressed on B and T lymphocytes. Different from normal B cells that only transiently express CD70 upon activation, CLL B cells stably express the molecule at relatively high levels, suggesting a fundamental biologic abnormality and adding another example of the dysregulation of the immune system in CLL. Here we investigated the role of CD70 in CLL using blocking anti-CD70 mAbs in preclinical mouse models of the disease. We first confirmed stable expression of CD27 and CD70 in patient samples as well as on murine TCL1 CLL B cells, including TCL1-192 cells, a set of primary murine leukemic cells within which > 95% cells react to autoantigen stimulation via the B-cell receptor (BCR). Comparing with healthy BM, we found that primary CLL patient B cells express significantly higher levels of the CD27 - CD70 receptor-ligand pair at both mRNA and protein levels, as do TCL-1 mice. We next investigated the effects of an anti-mouse CD70 effector function silenced/blocking mAb, FcD-FR70, on TCL1-192 CLL cells in vitro and in vivo in preclinical models. In vitro, treating TCL1 CLL cells with FcD-FR70 significantly inhibited CD40L-induced CLL cell growth. In vivo, twice weekly 10 mg/kg treatments with FcD-FR70 antibody significantly reduced the number of murine CLL B cells in spleens, lymph nodes and blood. The levels of reduction were comparable to those observed in anti-CD19 mAb or ibrutinib treated mice. There was a significantly better treatment outcome when therapy was initiated earlier. Moreover, the level of soluble mouse CD27, the product of CD27 cleavage after interaction with CD70, was significantly reduced in sera of animals treated with FcD-FR70. Notably, FcD-FR70, but not anti-CD19 mAb or ibrutinib, significantly reduced CD70 membrane levels. CD27 expression levels were not altered by all treatments. As CD27 and CD70 are inversely expressed in B cells, due to tight control at the transcriptional and posttranslational levels, this result strongly suggests preferential depletion of malignant cells with the high CD70 expression, which also affects levels of soluble CD27. We then performed bulk RNAseq analysis in CD19+CD5+ splenic cells from mice treated with control or FcD-FR70 antibody. Genes differing by >1.5 fold (P < 0.01) by FcD-FR70 treatment between the two groups included upregulated cell cycle regulator cyren and interferon gamma inducible protein IfI211, as well as downregulated IL10, CCR7, PIM3, PIM1, TGFb3, TNFaip3, JunB and CD69. Importantly, IPA analysis of the altered genes suggested inhibition of TLR7, TLR9, TNF, NFKB, IL4, BCR, BTK, and CD40L signaling pathways. Thus, FcD-FR70 treatment blocked key survival pathways, inducing death of CLL B cells. Finally, we studied the effect of anti-CD70 antibodies on CLL B-T cell interactions. Adding the FcD-FR70 mAb to an allo-MLR significantly blocked T cell proliferation and survival induced by TCL1-192 leukemic B cells (P < 0.01). We then tested the effects of 41D12-FcD, a blocking mAb in human IgG1 format that binds with picomolar affinity to human CD70, in a primary CLL patient derived xenograft model. This revealed a significant reduction of malignant cells in the spleen, BM, and peritoneum (P < 0.001). Interestingly, the efficacy of the blocking 41D12-FcD antibody was associated with a significant decrease of human T cells, suggesting that the antibody directly blocked the proliferation of malignant B cells and also inhibited T cell-CLL cell interactions. Notably, no significant changes were seen in the differentiation of T cells, expression of CTLA4, PD1, PD-L1, and levels of Th1/Th2 cytokines after anti-CD70 treatment. Overall, data from our preclinical models suggest targeting CD70 in CLL alters BCR, CD40L, IL4, and TNF receptor signaling and delays CLL disease progression. Although the involvement of CD70 and the relationships between CLL B cells and non-malignant cells needs further study, the availability of clinical stage therapeutic anti-CD70 antibodies supports the use anti-CD70 immunotherapy in combination therapies in CLL.

CLL-506 Clinical Outcomes Beyond Progression on Ibrutinib in Patients With Chronic Lymphocytic Leukemia

Patients with chronic lymphocytic leukemia (CLL) with disease progression on ibrutinib have worse outcomes compared to patients stopping ibrutinib due to toxicity. To analyze survival estimates of patients with CLL following true disease progression (per iwCLL 2018 criteria) on ibrutinib. Retrospective. Academic. Patients with CLL who received ibrutinib therapy for CLL at a Mayo Clinic Cancer Center site (Arizona, Florida, Minnesota) between 4/2012-6/2021. Treatment-free survival (TFS) was analyzed as the duration from the start of treatment immediately after ibrutinib failure to the start of the subsequent line of therapy or death, whichever occurred earlier. Overall survival (OS) was analyzed as the time from date of progression while on ibrutinib and from subsequent therapy start date until date of death or last known to be alive. OS was analyzed using the Kaplan-Meier method (with comparisons of OS by characteristics analyzed by the log rank test). Among 144 patients with CLL identified to have disease progression on ibrutinib, the median age was 68 years and 74% were male. Unmutated IGHV (90%) and TP53 disruption (50%) were common in evaluable patients at the time of progression on ibrutinib. The median OS for the entire cohort following progression on ibrutinib was 25.5 months; 29.8 months and 8.3 months following CLL progression (n=104) and Richter's transformation (n=38), respectively. Longer OS was observed among patients with CLL progression who had received ibrutinib in the frontline (n=23) compared to relapsed/refractory setting (not reached versus 28.5 months; p=0.04), but was similar amongst patients treated with 1, 2, or ≥3 prior lines (18.5, 30.9, and 26.0 months, respectively, p=0.24). Among patients with CLL disease progression on ibrutinib, next-line treatment with chimeric antigen receptor T-cell therapy or venetoclax-based treatment was associated with significantly longer median OS and TFS compared to other approved treatments (OS: not-reached, 29.8 months, and 9.1 months, respectively, p=0.034; TFS: 30.4 months, 20.1 months, and 4.4 months, respectively, p<0.001) Conclusions: These findings suggest an unmet need for this growing patient population while supporting the current practice of next-line venetoclax and participation in clinical trials.

Clinical outcomes in patients with chronic lymphocytic leukemia with disease progression on ibrutinib

Patients with chronic lymphocytic leukemia (CLL) with disease progression on ibrutinib have worse outcomes compared to patients stopping ibrutinib due to toxicity. A better understanding of expected outcomes in these patients is necessary to establish a benchmark for evaluating novel agents currently available and in development. We evaluated outcomes of 144 patients with CLL treated at Mayo Clinic with 2018 iwCLL disease progression on ibrutinib. The median overall survival (OS) for the entire cohort was 25.5 months; it was 29.8 months and 8.3 months among patients with CLL progression (n = 104) and Richter transformation (n = 38), respectively. Longer OS was observed among patients with CLL progression who had received ibrutinib in the frontline compared to relapsed/refractory setting (not reached versus 28.5 months; p = 0.04), but was similar amongst patients treated with 1, 2, or ≥3 prior lines (18.5, 30.9, and 26.0 months, respectively, p = 0.24). Among patients with CLL disease progression on ibrutinib, OS was significantly longer when next-line treatment was chimeric antigen receptor T-cell therapy (median not reached) or venetoclax-based treatment (median 29.8 months) compared to other approved treatments, such as chemoimmunotherapy, phosphoinositide 3’-kinase inhibitors, and anti-CD20 monoclonal antibodies (9.1 months; p = 0.03). These findings suggest an unmet need for this growing patient population.

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

AbstractThe BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter’s syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Lack of Bmf Facilitates the Selection of Highly Responsive B-Cell Receptor Clones in Chronic Lymphocytic Leukemia

Introduction: Chronic lymphocytic leukemia (CLL) is a disease of inhibited cell death, increased proliferation and high importance of interactions with the microenvironment e.g. with T-cells or stromal cells. A central effector in this concert is the activation of B-cell receptor (BCR) signalling pathway, associated with selection of specific BCR qualities (either autonomous signalling or reactivity to chronic (auto)-antigenic stimuli) and these signals play an essential role in CLL disease development and progression, also evidenced by the clinical success of kinase inbibitors directed at this signal. Mechanisms modulating selection of specific BCR types are ill understood, but since checkpoints during BCR selection in B cells are guarded by the Bcl2 family of proteins it is likely that cell death proteins are also influencing these decisions in CLL. To study the importance of microenvironmental interactions and antigen stimulation Tcl1 tg mice, which develop a murine CLL highly similar to the human disease 1,2, were frequently applied in the past, aiming to overcome the limitations of more or less artificial CLL in vitro culture systems. During clonal evolution of CLL, predominantly unmutated and stereotyped IgVH-11 and IgVH-12 BCRs are selected in Tcl1 tg mice which were shown to be specific for the autoantigen phosphatidylcholine (PtC) 3, however, the detailed mechanisms of clonal selection in CLL are still unclear. Here we propose a role of the BH3-only and pro-apoptotic protein Bmf in clonal selection by eliminating CLL cells expressing highly responsive BCRs.Methods: Tcl1 tg mice were crossed to Bmf knockout (KO) mice and CLL development monitored over time. After establishment of overt leukemia, mice were sacrificed and the overall survival time analysed. Additionally, we performed BCR sequencing, RNA-Seq and phosphoproteomics after 10µg/ml aIgM treatment using mass cytometry (Helios, CyTOF) and flow cytometry.Results: We found that Tcl1 tg Bmf KO mice developed phenotypically unchanged murine CLL but had an earlier onset of disease (Figure 1A) and an altered usage of BCR IgVH genes. While Tcl1 tg mice usually use PtC specific IgVH genes from the VH11 and V12 family, absence of Bmf led to a skewing to non PtC specific IgVH genes, mainly form the VH1 family (Figure 1B). We thus speculated that lack of Bmf favoured the selection of CLL clones with hyper responsive BCRs, when compared to Tcl1 tg mice with functional Bmf. Indeed we could show that after aIgM stimulation Tcl1 tg BmfKO CLL cells showed increased BCR signalling as measured by mass cytometry (Figure 1C), which was also confirmed by conventional flow cytometry (Figure 1D). Importantly, strong BCR stimulation induces cell death in B cells. Indeed, this can be observed in CLL cells from Tcl1 mice. In Tcl1 tumors that are deficient for BMF this cell death induction is not observed, suggesting that loss of Bmf protects from excessive cell death due to strong BCR stimulation.Conclusions: Here we report a novel role of Bmf in the selection of BCR clones in murine CLL. The higher BCR signalling capacity and the skewing of IgVH gene usage in the absence of Bmf indicates that highly responsive BCR clones are more often selected instead of eliminated during clonal selection.. Interestingly, a SNP in the Bmf gene has been reported to be susceptibility locud for CLL, suggesting that low expression of BMF may have similar effects in huma CLL. Indeed, in preliminary analyses, we observed a similar contribution of Bmf in clonal selection and BCR responsiveness in human CLL and decreased resistance to the BCR signalling inhibitor ibrutinib. Therefore our findings might also be important in a clinical context.

CXCL13 plasma levels function as a biomarker for disease activity in patients with chronic lymphocytic leukemia.

The chemoattractant CXCL13 organizes the cellular architecture of B-cell follicles and germinal centers. During adaptive immune responses, CXCL13 plasma concentrations transiently increase and function as a biomarker for normal germinal center activity. Chronic lymphocytic leukemia (CLL) cells express high levels of CXCR5, the receptor for CXCL13, and proliferate in pseudofollicles within secondary lymphoid organs (SLO). Given the morphologic and functional similarities between normal and CLL B-cell expansion in SLO, we hypothesized that CXCL13 plasma concentrations would correlate with CLL disease activity and progression. We analyzed CXCL13 plasma concentrations in 400 CLL patients and correlated the findings with other prognostic markers, time to treatment (TTT), CCL3 and CCL4 plasma concentrations, and in vivo CLL cell proliferation. We found that CXCL13 plasma concentrations were higher in CLL patients with active and advanced stage disease, resulting in a significantly shorter TTT. Accordingly, high CXCL13 levels correlated with other markers of disease activity and CCL3 levels. Higher CLL cell birth rates in vivo also associated with higher CXCL13 plasma concentrations. Interestingly, elevated CXCL13 plasma levels normalized during ibrutinib therapy, and increased in ibrutinib resistance patients. Collectively, these studies emphasize the importance of CXCL13 in crosstalk between CLL cells and the SLO microenvironment.

CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression

AbstractCD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d− subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d− cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

Increased frequency of CD4+ PD-1+ HLA-DR+ T cells is associated with disease progression in CLL.

Chronic lymphocytic leukaemia (CLL) patients often have abnormal expansions of CD4+ and CD8+ T cells and this can be associated with progressive disease. To characterise the key T-cell populations involved in this phenomenon, we used flow cytometry and 11 phenotypic markers to study 74 CLL patients and 14 controls. T cells of CLL patients were more phenotypically complex than those of healthy controls with significant increases in the frequencies of CD4 and CD8 memory T cells expressing exhaustion-, activation- and senescence-associated markers. Multivariate analysis of 111 different T-cell subsets showed that high frequencies of four subsets (three CD8 and one CD4) were associated with shorter progression-free survival. The most significant association was with CD4+ HLA-DR+ PD-1+ T cells, and patients could be stratified into high- and low-risk groups based on the frequency of these T cells. The expansion of this CD4+ subset could not be accounted for by age, cytomegalovirus infection or increases in Treg cells. Overall, these results highlight two relatively simple biomarkers, percentage CD8+ and percentage CD4+ PD-1+ HLA-DR+ T cells, which can be used to risk-stratify CLL patients, independent of other tumour-associated markers. They also provide further evidence for the pivotal role of T cells in modulating the pathology of CLL.

BIRC3 Expression Predicts CLL Progression and Defines Treatment Sensitivity via Enhanced NF-κB Nuclear Translocation.

Chronic lymphocytic leukemia (CLL) pathophysiology is characterized by a complex crosstalk of tumor cells with the microenvironment. In this regard, NF-κB signaling is considered as important signaling axis, with a variety of key molecules aberrantly expressed or genetically altered in patients with CLL. One of these molecules is BIRC3 (cIAP2), a central regulator of noncanonical NF-κB signaling that serves as pathway brake in the absence of microenvironmental signals. However, the contribution of BIRC3 expression to CLL progression and potential therapeutic implications is unknown.Experimental Design: We analyzed the role of BIRC3 mRNA expression in primary CLL samples in correlation to clinical datasets and used ex vivo assays to investigate functional consequences on the level of NF-κB signaling and downstream target gene regulation. For proof-of-principle experiments, we used genetically modified cell lines. We demonstrate that patients with CLL with low BIRC3 expression experience a more rapid disease progression, which coincides with an enhanced activation of canonical NF-κB target genes evidenced by an increased p65/Rel-B nuclear translocation ratio. As a consequence of enhanced canonical NF-κB target gene activation, both anti- and proapoptotic Bcl-2 family members were upregulated in BIRC3low primary CLL cells, which was associated with higher sensitivity to venetoclax treatment in vitro. Here we show the impact of BIRC3 expression in CLL disease progression in the absence of BIRC3 mutations and show altered canonical NF-κB target gene activation with therapeutic implications.

Psoas Abscess Due to Mycobacterium avium in A Patient with Chronic Lymphocytic Leukemia－Case Report and Review.

Infections may constitute a serious complication in patients with chronic lymphocytic leukemia (CLL). New treatment agents including obinutuzumab and ibrutinib have improved the progression-free survival in CLL, and data suggest a similar overall infection risk and a limited risk of opportunistic infections when compared to standard chemo-immunotherapy. Nevertheless, cases of opportunistic infections including non-tuberculous mycobacterial (NTM) in CLL patients have recently been published. We present a case of a 74-year old man with extensive prior CLL treatment history, including most recently obinutuzumab. He developed an abscess of the psoas muscle and inguinal lymphadenopathy. An inguinal node biopsy specimen showed infection with Mycobacterium avium, confirmed by broad-spectrum mycobacterial PCR, M. avium-specific PCR, and mycobacterial culture. This case and our literature review suggest that physicians should be aware of opportunistic infections in patients with CLL. Diagnostic differentiation from CLL disease progression, Richter’s transformation to aggressive lymphoma, and secondary malignancy relies on histological and appropriate microbiological studies from biopsy material of affected organs. Infection prophylaxis in CLL should be considered, including vaccinations and intravenous immune globulin replacement.

Research Advances of Monocyte/Macrophage in Chronic Lymphocytic Leukemia--Review

Chronic lymphocytic leukemia（CLL）is a malignant hematologic disease with heterogeneous clinical course, about two thirds of patients exhibit a long survival, but the remaining third of patients show aggressiveness of disease or poor response to conventional treatment even drug resistance. Studies suggested that tumor microenvironment possesses a critical effect in CLL disease progression in recent years. Monocyte/macrophage, as animportantcomponensof tumor microenvironment, play a promotive role in the growth and drug reistance of tumor cells by direct cell-to-cell contact, secreting cytokines and suppressing antitumor responses. Therefore, exploring the monocyte/macrophage in CLL can provide theoreticbasis for target treatment of CLL. In this review, the function of monocyte/macrophage and recent advances in therapy of CLL are discussed.