Hallmark Of Chronic Lymphocytic Leukemia Research Articles

Long-term immune changes in patients with relapsed/refractory chronic lymphocytic leukemia following treatment with venetoclax plus rituximab.

Immune dysregulation is a hallmark of chronic lymphocytic leukemia (CLL). Anti-CD20 antibodies (e.g., rituximab [R]) can be combined with venetoclax (Ven) to treat CLL. However, anti-CD20 antibodies can increase hypogammaglobulinemia risk, while the effects of Ven on immune dysregulation are still uncertain. We report long-term immune changes in VenR- and bendamustine-R (BR)-treated patients with relapsed/refractory CLL in the MURANO trial (NCT02005471). Patients were randomized to fixed-duration VenR (2 years Ven; VenR for the first 6 months) or BR (6 months). Immune cell levels were evaluated at the end of combination treatment (EOCT), end of treatment (EOT; VenR arm only), and 12 and 24 months post-EOCT. Overall, 130/194 VenR- and 134/195 BR-treated patients completed treatment without progressive disease. In patients who completed VenR combination therapy, median immunoglobulin (Ig)G and IgM levels decreased from baseline to EOT (p ≤ 0.01 and p ≤ 0.0001, respectively); by 24 months, post-EOT IgG had returned to baseline level and IgM had increased from baseline (p ≤ 0.001). Median IgA levels increased from baseline to 12 (p ≤ 0.0001) and 24 months post-EOT (p ≤ 0.0001). In BR-treated patients, changes in IgG, IgA, and IgM levels across the assessed time points were not significant, and by 24 months, post-EOCT IgG, IgA, and IgM were above baseline levels. Grade ≥3 infection rates on treatment were low. Overall, immune recovery was observed with VenR and BR, with stabilization of Ig levels after treatment. Post-treatment infection rates were generally low, making these very tolerable therapies for CLL.

P66Shc deficiency in CLL cells enhances PD-L1 expression and suppresses immune synapse formation.

Introduction: Escape from immunosurveillance is a hallmark of chronic lymphocytic leukemia (CLL) cells. In the protective niche of lymphoid organs, leukemic cells suppress the ability of T lymphocytes to form the immune synapse (IS), thereby hampering T-cell mediated anti-tumoral activities. By binding its cognate receptor PD-1 at the surface of T lymphocytes, the inhibitory ligand PD-L1, which is overexpressed in CLL cells, mediates the T-cell suppressive activities of CLL cells. However, the molecular mechanism underlying PD-L1 overexpression in CLL cells remains unknown. We have previously reported a defective expression of the pro-apoptotic and pro-oxidant adaptor p66Shc in CLL cells, which is causally related to an impairment in intracellular reactive oxygen species (ROS) production and to the activation of the ROS-sensitive transcription factor NF-κB. The fact that PD-L1 expression is regulated by NF-κB suggests a mechanistic relationship between p66Shc deficiency and PD-L1 overexpression in CLL cells. Methods: 62 treatment-naive CLL patients and 43 healthy donors were included in this study. PD-L1 and p66Shc expression was quantified in B cells by flow cytometry and qRT-PCR. IS architecture and local signaling was assessed by flow cytometry and confocal microscopy. CD8+ cell killing activity was assessed by flow cytometry. Results: Here we show that residual p66Shc expression in leukemic cells isolated both from CLL patients and from the CLL mouse model Eμ-TCL1 inversely correlated with PD-L1 expression. We also show that the PD-L1 increase prevented leukemic cells from forming ISs with T lymphocytes. Reconstitution of p66Shc, but not of a ROS-defective mutant, in both CLL cells and the CLL-derived cell line MEC-1, enhanced intracellular ROS and decreased PD-L1 expression. Similar results were obtained following treatment of CLL cells with H2O2 as exogenous source of ROS, that normalized PD-L1 expression and recovered IS formation. Discussion: Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to enhance PD-L1 expression and provides a mechanistic basis for the suppression of T cell-mediated anti-tumoral functions in the immunosuppressive lymphoid niche.

Long-Term Host Immune Changes Following Treatment with Venetoclax Plus Rituximab in Relapsed/Refractory Chronic Lymphocytic Leukemia

Background: Immune dysregulation is a hallmark of chronic lymphocytic leukemia (CLL) and often leads to hypogammaglobulinemia and frequent infections. Treatment with anti-CD20 antibodies (Abs) such as rituximab (R) is a known risk factor for hypogammaglobulinemia. Despite the widespread use of anti-CD20 Abs in combination with venetoclax (Ven), a B cell lymphoma 2 inhibitor, little has been reported on the long-term impact of this therapy on B cell recovery. We report long-term immune changes post treatment in patients (pts) with relapsed/refractory CLL in the MURANO phase 3 trial (NCT02005471) who completed the full VenR course without progressive disease (PD). Methods: Overall, 389 pts were randomized to either VenR (2 years of Ven with R for the first 6 months) or 6 months of bendamustine-R. Minimal residual disease (MRD) status has been previously reported (Kater et al. J Clin Oncol 2020). In the VenR arm, immunoglobulin (Ig) G, A and M, and T, B, and natural killer (NK) cells were measured centrally at baseline (BL), cycle 4 day 1 (C4D1), end of combination treatment (EOCT), end of treatment (EOT), and every 3 months for up to 3 years, then every 6 months until PD. This is a post hoc analysis among evaluable pts; statistics are descriptive only. Results: In total, 130 pts completed 2 years of VenR without PD (27 pts were excluded from this analysis as they received Ig therapy during the study). BL characteristics amongst those included were consistent with the whole population. Median change from BL by visit (EOT, 12 months, and 24 months post EOT) was calculated among pts with paired evaluable BL and visit samples (Table 1). IgG was significantly decreased at EOT vs BL (n=91; median change from BL, -5.9%). At 12 months (n=77; median change from BL, -4.8%) and 24 months (n=58; median change from BL, 1.1%) post treatment, IgG was not significantly different to BL levels. While there was no reduction in IgA from BL at EOT, at 12 and 24 months post treatment, IgA levels were significantly increased (median change from BL, 12.5% and 35.1%, respectively). IgM levels were significantly reduced at EOT vs BL (median change from BL, -32.3%), but returned to BL levels at 12 months post treatment (median change from BL, 11.1%). At 24 months post treatment, IgM had significantly increased by 49.5% compared with BL levels. There was no significant difference in median IgG, IgA and IgM levels at any time point amongst pts who achieved undetectable (u)MRD at EOT vs pts with MRD at EOT. In pts who received VenR the overall infection rate was low; however, there was a non-statistically significant, but numerically higher rate of grade ≥3 infections occurring on treatment in those with uMRD at EOT compared with those who had MRD at EOT (15.2% [10/66] vs 7.1% [2/28]; p=0.5). T, B and NK cell data were available for all 130 pts. A depletion in CD19+ B cells was observed on treatment, which was more pronounced in pts with uMRD at EOT. After EOCT, B cell recovery started ~1 year later, and stabilized ~30 months after EOCT, regardless of EOT MRD status (Figure 1). Given the differences in disease burden at EOT, comparable CD19+ B cell recovery in pts with uMRD and MRD is likely to be representative of a larger relative proportion of normal B cells vs CLL cells. Additional analyses to test this are underway. At BL, total CD3+ T cells were increased (median [range], 1,991 [135-17,334]; reference values as previously reported, 700-2,100/µL [de Weerdt et al. Blood Adv 2019]). Though a reduction of CD3+ T cells was observed during combination treatment (C4D1), the number of CD3+ T cells were maintained within the reference range post treatment. A similar pattern was observed for NK cells. The median (range) CD4/CD8 ratio at BL was 1.10 (0.33-5.77) and was maintained throughout the study. Conclusions: Overall, pts demonstrated immune recovery following fixed-duration treatment with VenR in this post hoc exploratory analysis. This is the first time that post-treatment serial Ig levels have been reported following VenR therapy. Recovery of IgG, IgA and IgM levels post treatment was observed regardless of EOT MRD status. While the overall incidence of grade ≥3 infections was low, the rate was numerically higher in pts with uMRD at EOT compared with pts with MRD. Additional analyses are underway to assess the correlation between infection and MRD state. A normalization of CD3+ T cells during and after VenR is consistent with previous reports. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tumor Heterogeneity-Based Sscr-Score Predicts Prognosis and Reveals Mechanism of SORL1 in Restraining CLL Progression

Introduction Complex heterogeneity is a hallmark of chronic lymphocytic leukemia (CLL) that challenges oncological research and optimal treatment regimens. Although genetic markers with prognostic values have been developed for risk assessment in CLL, the existing prognostic models are far from satisfaction. Single-cell RNA sequencing (scRNA-seq) technologies have made it possible to illustrate the genetic heterogeneity and cell differentiation status at the single-cell level. A prognostic model associated with CLL cell differentiation status was established to refine the risk assessment of CLL patients. Methods Primary CLL cells from 90 patients and CD19+ B cells from 20 healthy donors were collected with informed consents according to the Declaration of Helsinki. Expression levels of SORL1 mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Cell viability was measured by the CCK-8 assay. Cell cycle and apoptosis were analyzed by PI and Annexin V-PE/7AAD staining. Fatty acid oxidation rate was assessed with Fatty Acid Oxidation Complete Assay Kit. Red oil O staining was performed to evaluate the lipid deposition in CLL cells. Single cell sequencing data were analyzed by the Seurat package and Monocle package in R4.0.5. Results The scRNA-seq profile revealed remarkable intra- and inter-tumoral heterogeneity of CLL. According to the differentiation status, this study reconstructed the evolutionary history of CLL cells, which reflected the molecular classification and risk subgroups of CLL. At the key point of cell differentiation, CLL cells were divided into two subsets with progressive and indolent genetic signatures respectively. Genes differentially expressed between aggressive and indolent subsets were identified. Through univariate and multivariate analysis, four genes (SORL1, SGK1, CYBB, RPL22L1) with significant prognostic values were selected to construct a risk model named SSCR-Score. The risk score was calculated and validated in two independent validation cohorts (Fig 1A). SSCR-Score has revealed the positive prognostic value of SORL1 in CLL. Increased expression of SORL1 was associated with prolonged overall survival and improved time to first treatment in CLL. Among patients with ZAP70 (-) or IGHV mutated status, the expression levels of SORL1 were up-regulated. Although increased expression of SORL1 was associated with favorable prognosis, the biological mechanism of SORL1 still remained ill-defined. This study demonstrated that overexpression of SORL1 restrained cell proliferation, induced cell apoptosis and G2/M cell cycle arrest in CLL cells. In accordance with the RNA sequencing profile, overexpression of SORL1 suppressed abnormal lipid reposition and fatty acid oxidation in CLL cells. On a molecular level, SORL1 down-regulated the expression of lipid-metabolism related genes, including lipoprotein lipase (LPL), carnitine palmitoyltransferase 1A (CPT1A) and carnitine palmitoyltransferase 2 (CPT2). To illustrate the SORL1-mediated regulation of LPL, immunofluorescence staining was performed. Results showed that SORL1 accelerated the degradation of LPL. After the inhibition of lysosome function, accelerated degradation of LPL was not observed in CLL cells overexpressing SORL1. Rescue experiments substantiated that SORL1 impeded progression of CLL through downregulating the activity of LPL which promoted lipid metabolism. Immuno-coprecipitation experiment showed that anti-SORL1 antibody precipitated LPL protein. Additionally, 6-Shohaol induced overexpression of SORL1 in CLL cells. It exhibited potent anti-CLL efficacy by suppressing cell proliferation, promoting cell apoptosis and G2/M cell cycle arrest in CLL cells (Fig 1B). Conclusion CLL presented remarkable heterogeneity which reflected different biological subclasses and risk groups. Through identifying genes differentially expressed between aggressive and indolent cell subsets, a novel prognostic model named SSCR-Score was developed to optimize risk stratification of CLL patients. Moreover, SORL1 restrained progression of CLL by inhibiting lipid metabolism regulated by LPL in CLL cells. SORL1 may serve as a potential strategy for the development of novel therapeutic target in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TGF-β/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression.

Simple SummaryTGF-β is a key immunoregulatory pathway that can limit the proliferation of B-lymphocytes. Chronic lymphocytic leukemia (CLL) has been historically conceptualized as a neoplasm characterized by accumulation of mature B cells escaping programmed cell death and undergoing cell-cycle arrest in the G0/G1 phase. However, new evidence indicates that tumor expansion is in fact a dynamic process in which cell proliferation also plays an important role. In general, cancers progress by the emergence of subclones with genomic aberrations distinct from the initial tumor. Often, these subclones are selected for advantages in cell survival and/or growth. Here, we provide novel evidence to explain, at least in part, the origins of CLL progression in a subgroup of patients with a poor clinical outcome. In this cohort, the immunoregulatory pathway TGF-β/SMAD is modulated by miR-26b-5p and the impairment of this axis bypasses cell cycle arrest in CLL cells facilitating disease progression.Clinical and molecular heterogeneity are hallmarks of chronic lymphocytic leukemia (CLL), a neoplasm characterized by accumulation of mature and clonal long-lived CD5 + B-lymphocytes. Mutational status of the IgHV gene of leukemic clones is a powerful prognostic tool in CLL, and it is well established that unmutated CLLs (U-CLLs) have worse evolution than mutated cases. Nevertheless, progression and treatment requirement of patients can evolve independently from the mutational status. Microenvironment signaling or epigenetic changes partially explain this different behavior. Thus, we think that detailed characterization of the miRNAs landscape from patients with different clinical evolution could facilitate the understanding of this heterogeneity. Since miRNAs are key players in leukemia pathogenesis and evolution, we aim to better characterize different CLL behaviors by comparing the miRNome of clinically progressive U-CLLs vs. stable U-CLLs. Our data show up-regulation of miR-26b-5p, miR-106b-5p, and miR-142-5p in progressive cases and indicate a key role for miR-26b-5p during CLL progression. Specifically, up-regulation of miR-26b-5p in CLL cells blocks TGF-β/SMAD pathway by down-modulation of SMAD-4, resulting in lower expression of p21−Cip1 kinase inhibitor and higher expression of c-Myc oncogene. This work describes a new molecular mechanism linking CLL progression with TGF-β modulation and proposes an alternative strategy to explore in CLL therapy.

METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL

METTL3 Dysregulates RNA Splicing by Translational Control of Splicing Factors via m 6A Modification in CLL

The multi-kinase inhibitor TG02 induces apoptosis and blocks B-cell receptor signaling in chronic lymphocytic leukemia through dual mechanisms of action

The constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

Humoral Dysfunction in Patients with Chronic Lymphocytic Leukemia on Ibrutinib As Evidenced By Suboptimal Vaccine Responses

Background: Humoral and cell-mediated immune dysregulation is a hallmark of chronic lymphocytic leukemia (CLL) and predisposes these patients to recurrent infections, which remain the leading cause of mortality. Ibrutinib is widely used in both treatment-naive and relapsed CLL, and these patientshave a 48% cumulative rate of ≥grade 3 infections over a 5-year follow up (O'Brien et al.; Blood, April 2018). Our study evaluated humoral immunity in patients with CLL treated with ibrutinib by checking immunoglobulin (Ig) levels and vaccine responses. Methods: We measured serum IgG, IgM, IgA, and pre/post-vaccine IgG titers to diphtheria, tetanus, and Streptococcus pneumoniae in patients with CLL on treatment with ibrutinib. Patients were vaccinated with tetanus/diphtheria (Td) and the pneumococcal polysaccharide (PPV23) vaccines, with post-vaccination titers measured at four weeks. Descriptive statistics are reported with medians and interquartile ranges (IQR). Response to Td was defined as a two-fold increase in specific IgG into the protective range. Response to PPV23 was defined as a two-fold increase from baseline if pre-vaccination specific IgG ≥1.3 microgram per milliliter (mcg/ml), or if titers increased by four-fold from baseline if pre-vaccination specific IgG < 1.3 mcg/ml, for > 70% of the serotypes. Results: Ten patients were enrolled. The median age was 65 (IQR: 60-68) years; seven were male. The median time since diagnosis was 4.7 (IQR: 4-9.1) years. Patients were on ibrutinib for a median time of 1.3 (IQR: 0.4-2.8) years at the time of enrollment. Three patients had previously received treatment with rituximab monotherapy, while two patients had received prior combination therapy with fludarabine-rituximab-lenalidomide and fludarabine-cyclophosphamide-rituximab. Nine patients had received at least one course of antibiotics (ABx) in the year before enrollment; the median number of ABx courses was 2 (IQR 2-3). Four patients were on prophylactic antibiotics, and three had previously received immunoglobulin replacement therapy. The median serum IgG titer was 650 (IQR: 544-820) mg/dl. 40% (4/10) of patients responded to vaccination with diphtheria, 30% (3/10) of patients responded to vaccination with tetanus, and none of the patients responded to PPV23 (see Table 1). Conclusion: Patients with CLL treated with ibrutinib demonstrate a high prevalence of humoral dysfunction, as evidenced by suboptimal vaccine responses to both peptide and polysaccharide antigens despite adequate Ig levels. Clinicians should consider checking vaccine responses in conjunction with Ig levels for comprehensive evaluation of humoral dysfunction. Disclosures Mustafa: Genentech: Speakers Bureau; AstraZeneca: Speakers Bureau; Regeneron: Speakers Bureau; CSL Behring: Speakers Bureau. Jamshed:Takeda, Amgen and Celgene: Honoraria.

Functional Differences Between EBV- and CMV-Specific CD8+ T cells Demonstrate Heterogeneity of T cell Dysfunction in CLL.

Acquired T cell dysfunction is a hallmark of chronic lymphocytic leukemia (CLL), and is linked to an increased risk of infections, but also reduced immune surveillance and disappointing responses to autologous T cell-based immunotherapy. The mechanisms of T cell dysfunction in CLL are not well understood. Studying immunity against chronic viruses allows for detailed analysis of the effect of CLL on T cells chronically exposed to a specific antigen. Cytomegalovirus (CMV) reactivations are rare in CLL, which corroborates with preserved CMV-specific T cell function. Epstein-Barr virus (EBV) is another herpesvirus that results in chronic infection, but unlike CMV, is characterized by subclinical reactivations in CLL patients. Since both herpesviruses induce strong CD8+ T cell responses, but have different clinical outcomes, studying these specific T cells may shed light on the mechanisms of CLL-induced T cell dysfunction.We first analyzed the phenotype of EBV-specific CD8+ T cells in CLL and healthy controls, and found that in CLL EBV-specific CD8+ T cells are in an advanced differentiation state with higher expression of inhibitory receptors. Secondly, CLL-derived EBV-specific CD8+ T cells show reduced cytotoxic potential, in contrast to CMV-specific T cells. Finally, we performed transcriptome analysis to visualize differential modulation by CLL of these T cell subsets. While T cell activation and differentiation genes are unaffected, in EBV-specific T cells expression of genes involved in synapse formation and T cell exhaustion is altered. Our findings on the heterogeneity of antigen specific T cell function in CLL aids in understanding immune-dysregulation in this disease.

Response to the Shingrix Varicella Zoster Virus (VZV) Vaccine in Patients with Chronic Lymphocytic Leukemia (CLL) That Are Treatment Naive or Treated with a Bruton's Tyrosine Kinase Inhibitor (BTK-I)

Introduction Immune dysregulation is a hallmark of CLL, making these patients particularly vulnerable to infectious complications. Patients with CLL are at increased risk of developing varicella zoster virus (VZV) reactivation (shingles) due to their advanced age and immunocompromised status. A new recombinant (non-live) adjuvanted shingles vaccine (SHINGRIX; RZV) has the potential to reduce VZV reactivation, without the risk of giving a live vaccine to immunocompromised individuals. RZV is proven to reduce the risks of herpes zoster and postherpetic neuralgia in healthy adults ≥ 50 years of age, however its efficacy in immunocompromised individuals, including CLL, remains unknown. We report preliminary safety and efficacy of RZV in treated and untreated CLL patients. Methods In this phase II open-label study (NCT03702231), patients with CLL who were either treatment naïve or receiving treatment with a Bruton's tyrosine kinase inhibitor (BTK-I) (ibrutinib or acalabrutinib) received 2 doses of RZV via intramuscular injection at 0- and 3-months. Subjects were followed for 6 months and received assessment of serologic response at 3- and 6-months. Based on results of prior published studies, serologic response was defined as a ≥ four-fold rise in VZV anti-glycoprotein E (anti-gE) blood IgG serum titer after completing the RZV vaccine series. Additionally, serologic response was analyzed 3 months following the first vaccine administration to study the kinetics of the humoral vaccine response. All subjects completed an adverse event (AE) diary documenting any local (injection site) or systemic AE that started within 7 days after receiving the first and second vaccine dose. The data reported herein are as of July 12th 2019; updated results will be presented at the meeting. Results Safety data are available on 57 subjects who received at least one vaccine dose. The most frequent local and systemic AEs were injection site pain (67%), injection site reaction (32%) and generalized myalgias (25%) (Table 1). All adverse reactions were grade 1-2, except for 2 (4%) grade 3 reactions. No serious AEs were reported. All AEs resolved or returned to baseline within 7 days of vaccine administration. Seven subjects have completed the primary endpoint and have had serologic response assessment at 6-months. Serologic responses were observed in 3 (43%) patients. All patients (n = 7) achieved ≥ 2.2-fold rise in VZV anti-gE titers at 6-months. Preliminary analysis of 3-month samples (n = 43) shows early evidence of increasing titers after the first dose of RZV. Conclusions RZV administration appears to be safe in CLL patients that are treatment naïve or receiving treatment with a BTK-I. Compared to previously reported toxicities in the healthy population, no increase in frequency or severity of AEs were observed. Preliminary data suggests that RZV induces humoral immune responses in patients with CLL. Disclosures Wiestner: Pharmayclics: Research Funding; Acerta: Research Funding; Merck: Research Funding; Nurix: Research Funding.

STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia

BackgroundDysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study.MethodsAn extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients.ResultsSeeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup.ConclusionsThese data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

Abstract 1012: CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo

Abstract The hallmark of chronic lymphocytic leukemia (CLL) cells is their re-circulation between peripheral blood and immune niches to obtain pro-proliferative and pro-survival signals. CLL cells that have recently exited the immune niches to the peripheral blood are characterized by low cell-surface levels of chemokine receptor CXCR4 and high levels of activation molecule CD5. These CXCR4dimCD5bright CLL cells have a ~2-fold higher CD20 expression due to the activation of the CXCR4/SDF-1 axis (Pavlasova et al., Blood, 2016). We hypothesized that CD20 up-regulation in the context of a microenvironment is required for some functional regulation. We hypothesized that CD20 expression is of importance for B-cell receptor (BCR) signaling as we observed that CXCR4dimCD5brightCD20bright CLL cells have also ~2-fold higher surface IgM levels (P<0.0001). This was coupled with higher responsiveness to BCR-crosslinking with anti-IgM (P=0.0015). CD20 levels directly affect BCR-induced calcium flux and the phosphorylation of BCR/PI3K-associated molecules (LYN, SYK, ERK, GAB1) after BCR-crosslinking. The CXCR4dimCD5brightCD20bright subpopulation contains more proliferative (Ki67+) cells, higher levels of pAKT/pERK/pCD79a (P<0.001), and their gene expression signature (NGS Illumina) is significantly enriched for genes involved in BCR and MAPK signaling, migration, and actin cytoskeleton organization (P<0.0001). Finally, we have shown that rituximab primarily and potently eliminates the CXCR4dimCD5brightCD20bright CLL cells (P<0.0001). Overall, rituximab was ~9-fold more efficient in eliminating CXCR4dimCD5brightCD20bright CLL cells than CXCR4brightCD5dimCD20dim cells (P=0.03) during FCR therapy in vivo. Altogether, we described that higher CD20 expression supports BCR signaling and contributes to the activated phenotype and aggressiveness of an intra-clonal subpopulation of CXCR4dimCD5brightCD20bright cells. This is a first mechanistic explanation of CD20 function in CLL cells. Additionally, it is tempting to speculate that rituximab's clinical success is at least partially attributed to the preferential elimination of the intra-clonal proliferative subpopulation of BCR-proficient CLL cells. Supported by: the Ministry of Education, Youth and Sports of the Czech Rep. under the project CEITEC 2020 (LQ1601); Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Rep., grant No. 16-29622A. All rights reserved. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 692298. This study reflects only the author's view and the Research Executive Agency is not responsible for any use that may be made of the information it contains. MH CZ-DRO (FNBr, 65269705); MUNI/H/0865/2016; MUNI/A/0968/2017. Citation Format: Gabriela Pavlasova, Marek Borsky, Veronika Svobodova, Jan Oppelt, Katerina Cerna, Jitka Novotna, Katerina Musilova, Vaclav Seda, Eva Vojackova, Yvona Brychtova, Michael Doubek, Sarka Pospisilova, Jiri Mayer, Marek Mraz. CD20 supports BCR signaling in an intra-clonal aggressive chronic lymphocytic leukemia subpopulation of cells and rituximab primarily targets these BCR-proficient B cells in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1012.

Using anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in chronic lymphocytic leukemia (CLL)

Abstract Increased CD38 expression on chronic lymphocytic leukemia (CLL) cells correlates with a negative prognosis, however, its utility as a therapeutic target in this malignancy remains unknown. Our investigations show that the anti-CD38 therapeutic mAb, daratumumab (Dara) induces direct apoptosis of CLL cells and promotes their immune-mediated clearance (through ADCC). Notably, CD38 expression is quite high on a subset of CLL-cells (CD19+/CD38hi/CD24+/IL-10+; B-regulatory-like phenotype), which promote Treg expansion while prohibiting CD8+ cytotoxic T-cells (cTLs). Indeed, immunosuppression is a hallmark of CLL and patients often present with increased number of Tregs and Th2 cells but decreased effector T cells and cTLs. It has recently been reported that CD38hi Tregs are significantly immunosuppressive and when examining this population in peripheral blood mononuclear cells (PBMCs) from CLL patients (n=13), we noted that >50% of CD4+/CD25+/FoxP3+ cells were CD38hi (median MFI ~600). We hypothesized that Dara would kill CD38hi CLL cells à decreased CD38hi Treg formation/direct killing à expansion of cTLs. Indeed, ex-vivo treatment of CLL-patient derived PBMCs showed that Dara was highly lethal to both CD38hi “Breg-like” CLL cells and Tregs; associated with decrease in IL-10, increase in IFN-γ, effector T cells, Th17 subsets and proliferation of cTLs. Altogether this pointed towards recovery of anti-tumor immunity which was further evidenced by enhanced tumor-specific cytolytic activity of cTLs. Our pre-clinical data demonstrates that targeting CD38 in CLL has an anti-tumor effect via 1.) lethal effect on CLL-Bregs and Tregs and 2.) tumor-specific expansion of cTLs, which we will test clinically in an upcoming trial.

SCYLD expression leads to CLL in mice and men

Abstract A tightly regulated NF-κB pathway is very important for B cell homeostasis. Aberrant activation of NF-κB is involved in the development of chronic lymphocytic leukemia (CLL). The deubiquitinating enzyme CYLD, acts as a negative regulator of NF-κB by removing K63-linked ubiquitin chains from its target proteins. Here, we show that the B cell specific deletion of full-length CYLD and expression of the shorter splice form (sCYLD) lacking the binding sites for TRAF2 and NEMO, leads to the dramatic expansion of CD5 positive B cells, a hallmark of CLL. These cells accumulate in the blood and lymphatic organs with age, and show characteristic up-regulation of typical CLL-markers such as ZAP-70 and Bcl-2. A20, another negative regulator of NF-κB targets some of the same proteins as CYLD, thereby potentially compensating for the lack of FL-CYLD. We crossed sCYLD mice to a B cell specific A20 knockout. In these mice, the CLL phenotype progresses more rapidly probably due to an increase of NF-κB activity, up to the point where 100% of the B cells are CD5+. These cells show oligoclonal expansion, infiltrate the liver and the lungs, and accumulate in all lymphatic organs. Interestingly, we could show that some human CLL patients also express sCYLD, whereas none of the healthy controls do. These findings indicate that sCYLD is a new risk factor for CLL and implies that disrupted alternative splicing of key NF-κB regulators lead to the development of CLL.

Mechanisms of PD-L1/PD-1–mediated CD8 T-cell dysfunction in the context of aging-related immune defects in the Eµ-TCL1 CLL mouse model

AbstractT-cell defects, immune suppression, and poor antitumor immune responses are hallmarks of chronic lymphocytic leukemia (CLL), and PD-1/PD-L1 inhibitory signaling has emerged as a major immunosuppressive mechanism. However, the effect of different microenvironments and the confounding influence of aging are poorly understood. The current study uses the Eμ-TCL1 mouse model, which replicates human T-cell defects, as a preclinical platform to longitudinally examine patterns of T-cell dysfunction alongside developing CLL and in different microenvironments, with a focus on PD-1/PD-L1 interactions. The development of CLL was significantly associated with changes in T-cell phenotype across all organs and function. Although partly mirrored in aging wild-type mice, CLL-specific T-cell changes were identified. Murine CLL cells highly expressed PD-L1 and PD-L2 in all organs, with high PD-L1 expression in the spleen. CD3+CD8+ T cells from leukemic and aging healthy mice highly expressed PD-1, identifying aging as a confounder, but adoptive transfer experiments demonstrated CLL-specific PD-1 induction. Direct comparisons of PD-1 expression and function between aging CLL mice and controls identified PD-1+ T cells in CLL as a heterogeneous population with variable effector function. This is highly relevant for therapeutic targeting of CD8+ T cells, showing the potential of reprogramming and selective subset expansion to restore antitumor immunity.

Targeting Antiapoptotic and Proapoptotic Proteins for Novel Chronic Lymphocytic Leukemia Therapeutics

SUMMARY Deficiency in the apoptotic program is one of the hallmarks of chronic lymphocytic leukemia. Defective apoptosis mainly results from the constitutive activation of survival pathways, which leads to the transcription and overexpression of antiapoptotic factors. The latter include proteins of the Bcl-2 family and members of the IAP family. The strategy of inhibiting the expression or activity of these antiapoptotic factors has been extensively investigated. Conversely, upregulation of proapoptotic proteins, notably BH3-only members of the Bcl-2 family (capable of antagonizing their antiapoptotic counterparts) has also been consistently described. Either mechanism can promote apoptosis in chronic lymphocytic leukemia cells ex vivo. The present article recapitulates the mechanistic data and how they contribute to the development of therapeutic agents targeting apoptosis.

PIM Kinases Are Essential for Chronic Lymphocytic Leukemia Cell Survival (PIM2/3) and CXCR4-Mediated Microenvironmental Interactions (PIM1)

Overexpression of the CXCR4 receptor is a hallmark of chronic lymphocytic leukemia (CLL) and is important for CLL cell survival, migration, and interaction with their protective microenvironment. In acute myelogenous leukemia (AML), PIM1 was shown to regulate the surface expression of the CXCR4 receptor. Here, we show that PIM (proviral integration site for Moloney murine leukemia virus) kinases 1-3 are overexpressed and that the CXCR4 receptor is hyperphosphorylated on Ser339 in CLL compared with normal lymphocytes. Furthermore, CXCR4 phosphorylation correlates with PIM1 protein expression and PIM1 transcript levels in CLL. PIM kinase inhibition with three different PIM kinase inhibitors induced apoptosis in CLL cells independent of the presence of protective stromal cells. In addition, PIM inhibition caused dephosphorylation of the CXCR4 receptor on Ser339, resulting in enhanced ligand-dependent CXCR4 internalization and reduced re-externalization after withdrawal of CXCL12. Furthermore, PIM inhibition in CLL cells blocked CXCR4 functions, such as migration toward CXCL12- or CXCL12-induced extracellular signal-regulated kinase (ERK) phosphorylation. In concordance, pretreatment of CLL cells with PIM kinase inhibitors strongly reduced homing of CLL cells toward the bone marrow and the spleen of Rag2(-/-)γc(-/-) mice in vivo. Interestingly, the knockdown of PIM kinases in CLL cells demonstrated diverging functions, with PIM1 regulating CXCR4 surface expression and PIM2 and PIM3 as important for the survival of CLL cells. Our results show that PIM kinase inhibitors are an effective therapeutic option for CLL, not only by impairing PIM2/3-mediated CLL cell survival, but also by blocking the PIM1/CXCR4-mediated interaction of CLL cells with their protective microenvironment.

Monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic hematologic condition defined by the presence of a small (<5 x 10(9)/L) clonal B-cell population in the peripheral blood in the absence of lymph-node enlargement, cytopenias or autoimmune diseases. It is found in approximately 3-12% of normal persons depending on the accuracy of analytical techniques applied. According to the immunophenotypic profile of clonal B-cells, the majority of MBL cases (75%) are classified as chronic lymphocytic leukemia (CLL)-like. This form may progress into CLL at a rate of 1-2% per year. It is thought that CLL is always preceded by MBL. The remaining MBL cases are defined as atypical CLL-like (CD5+/CD20(bright)) and CD5(-) MBL. The MBL clone size is quite heterogenous. Accordingly, two forms of MBL are identified: i) high-count, or 'clinical' MBL, in which an evidence of lymphocytosis (<5 x 10(9)/L clonal B-cells) is seen, and ii) a low-count MBL, in which a normal leukocyte count is found and that is identified only in population-screening studies. Both forms of MBL may carry the cytogenetic abnormalities that are the hallmark of CLL, including 13q-, 17p- and trisomy 12. Consistent with the indolent phenotype of this condition, genetic lesions, such as TP53, ATM, NOTCH1 and SF3B1 mutations, usually associated with high-risk CLL, are rarely seen. Overall, no prognostic indicator of evolution of MBL to overt CLL has been found at present time. However, taking into account this possibility, a clinical and lab monitoring (at least annually), is recommended.

Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL. Disclosures:No relevant conflicts of interest to declare.

Abstract 2886: Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of Chronic Lymphocytic Leukemia B cells.

Abstract Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. To assess the aberrant behavior of BCR signaling in CLL B cells, we developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled the complex nature of BCR signaling using Partial Least Squares Regression (PLSR) to gain new insights into the strongest predictors of discrimination between CLL and normal B cells. We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. Furthermore, PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as key to the pathogenic properties of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Single-cell multidimensional analysis of BCR signaling permitted focused study of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness. Citation Format: M. Lia Palomba, Kelly Piersanti, Carly G. K. Ziegler, Jesse Cotari, Robert Balderas, Sami N. Malek, Erlene Seymour, Andrew D. Zelenetz, Marcel R. M. van den Brink, Gregoire Altan-Bonnet. Multidimensional single-cell analysis of BCR signaling reveals proximal activation defect as a hallmark of Chronic Lymphocytic Leukemia B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2013-2886 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.