Pathobiology Of 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.

Eμ-TCL1 adoptive transfer mouse model of chronic lymphocytic leukemia.

Despite being the most common adult leukemia in the western world, Chronic Lymphocytic Leukemia (CLL) remains a life-threatening and incurable disease. Efforts to develop new treatments are highly dependent on the availability of appropriate mouse models for pre-clinical testing. The Eμ-TCL1 mouse model is the most established pre-clinical approach to study CLL pathobiology and response to treatment, backed by numerous studies highlighting its resemblance to the most aggressive form of this malignancy. In contrast to the transgenic Eμ-TCL1 model, employing the adoptive transfer of Eμ-TCL1-derived splenocytes in immunocompetent C57BL/6 mice results in a comparably rapid (e.g., leukemic development within weeks compared to months in the transgenic model) and reliable model mimicking CLL. In this chapter, we would like to provide readers with a thoroughly optimized, detailed, and comprehensive protocol to use the adoptive transfer Eμ-TCL1 model in their research.

CD49d Expression Identifies a Biologically Distinct Subtype of Chronic Lymphocytic Leukemia with Inferior Progression Free Survival on BTK Inhibitor Therapy

Background: Acalabrutinib, a highly selective Bruton tyrosine kinase inhibitor (BTKi), is effective and well tolerated for patients with chronic lymphocytic leukemia (CLL). During treatment with BTKis, transient lymphocytosis is observed due to the efflux of CLL cells from lymphoid organs. CD49d, the alpha chain of the CD49d/CD29 integrin very late antigen-4 (VLA-4), is a key mediator in CLL microenvironmental interactions. High CD49d expression has been associated with inferior response to ibrutinib (Tissino, 2018). Here, we investigated the role of CD49d in response to acalabrutinib in 48 patients with CLL enrolled in a single-center, phase 2 study (NCT02337829). Methods: Patients were divided into CD49d positive (CD49d+, n = 27, 56%) and CD49d negative (CD49dneg, n = 21, 44%) groups based on the established 30% cutoff, as assessed by flow cytometry. CD49d activation was measured as VLA-4 receptor occupancy (RO), as described (Tissino, 2018). Time to progression was estimated using the Kaplan-Meier method. RNA sequencing (RNA-seq) of CD19+ selected CLL cells from CD49d+ (n = 10) and CD49dneg (n = 10) subsets was performed at baseline and 6 months on acalabrutinib. Results: After the first month on acalabrutinib, the median increase in absolute lymphocyte count (ALC) for CD49d+ CLL was 6.4% vs. 50.4% for CD49dneg (P = .02). However, the increase in ALC on day 3 of 42% and 48%, respectively, was comparable (P = .8). A subset of patients showed bimodal CD49d (bimCD49d) expression, with concomitant presence of CD49d positive and negative subpopulations. In these cases, the fraction of CD49d+ CLL cells increased significantly on day 3 (P = .002), but decreased at 6 months (P = .004), suggesting that CD49d+ cells may be better able to remain tissue bound on chronic therapy or reenter lymphoid tissues at higher rates than CD49dneg cells. While constitutive VLA-4 activation was reduced in patients on acalabrutinib, BCR and CXCR4 stimulation still increased VLA-4 RO on CLL cells even after 6 months of treatment. PI3K, in addition to BTK signaling, is implicated in inside-out activation of VLA-4. In vitro, addition of the PI3K inhibitor duvelisib to acalabrutinib-treated CLL cells completely blocked BCR- but not CXCR4- induced VLA-4 activation. The overall response rate of the study was 95.8% (Sun, 2020). Sixteen patients progressed at a median of 42.5 months, 3 with transformation. Time to progression for patients with CD49d+ and CD49dneg CLL (applying 30% cutoff) did not differ (P = .4). However, at 72 months 50% of the combined CD49d+ and bimCD49d patients (n = 37) were estimated progression-free vs 90% of patients with a homogeneous CD49dneg CLL population (n = 11, P = .02). Of 13 patients progressing with CLL, 12 (92%) were CD49d+ or bimodal CD49d and 11 (85%) had BTK and/or PLCG2 mutations. To dissect the impact of CD49d expression on CLL pathobiology and the response to acalabrutinib, we compared transcriptomes of CD49d+ and CD49dneg subsets by RNA-seq. A distinct gene expression profile separated the groups by principal component analysis. Using gene set enrichment analysis, CD49d+ CLL showed constitutively more immunoreceptor, NF-kappa B, and cytokine/JAK-STAT signaling, as well as enhanced cell survival, adhesion and migration capacity compared to CD49dneg CLL (FDR <.25). After 6 months on acalabrutinib, all 20 patients achieved a clinical response and all samples showed downregulation of B-cell receptor and NF-kappa B target genes, as expected for BTKi therapy. Interestingly, on acalabrutinib, CD49d+ samples not only maintained a higher level of activation compared to CD49dneg CLL, but differences in the degree of cytokine (IL-6, IL-10) and STAT3 signaling became more prominent. Conclusion. CD49d+ and CD49dneg CLL cells are rapidly mobilized out of lymph nodes at the start of acalabrutinib therapy, but CD49d+ cells appear able to reenter or persist in lymphoid tissues better than CD49dneg cells. Bimodal CD49dneg and CD49d+ cases share an increased risk of disease progression, with most patients having detectable BTK or PLCG2 mutations at the time of progression. The transcriptional program of CD49d+ CLL reflects upregulation of survival and activation pathways that may increase tolerance to BTKi therapy and set the stage for clonal evolution. Thus, CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance and a possible therapeutic target to improve on BTKi therapy.

Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation.

The indoleamine 2,3-dioxygenase 1 (IDO1) metabolic circuitry, comprising the first tryptophan (Trp) catabolite L-kynurenine (Kyn) and the aryl hydrocarbon receptor (AHR), has emerged as a mechanism of cancer immune evasion. Here, we investigated the functional role of the IDO1/Kyn/AHR axis in chronic lymphocytic leukemia (CLL). Our data show that CLL cells expressed an active form of the IDO1 enzyme and microenvironmental stimuli can positively modulate its expression. Interferon (IFN)-γ induces IDO1 expression through the Jak/STAT1 pathway and mediates Kyn production concomitantly with Trp consumption in CLL-conditioned media, while INCB018424 (ruxolitinib), a JAK1/2 inhibitor, impaired both effects. To characterize the involvement of IDO1 in leukemic cell maintenance, we overexpressed IDO1 by vector transfection measuring enhanced resistance to spontaneous apoptosis. IDO1 pro-survival influence was confirmed by treating CLL cells with Kyn, which mediated the increase of induced myeloid leukemia cell differentiation protein (MCL1). Conversely, AHR silencing or its blockade via CH-223191 improved the apoptosis of leukemic clones and mitigated MCL1 expression. Moreover, Kyn-treated CLL cells are less affected by the pro-apoptotic effect of ABT-199 (venetoclax), while CH-223191 showed synergistic/additive cytotoxicity with this drug. Lastly, targeting directly MCL1 in CLL cells with AMG-176, we abrogate the pro-survival effect of Kyn. In conclusion, our data identify IDO1/Kyn/AHR signaling as a new therapeutic target for CLL, describing for the first time its role in CLL pathobiology.

Targeting Mcl-1 by AMG-176 During Ibrutinib and Venetoclax Therapy in Chronic Lymphocytic Leukemia.

B-cell receptor (BCR) signaling pathway and Bcl-2 family prosurvival proteins, specifically Bcl-2 and Mcl-1, are functional in the pathobiology of chronic lymphocytic leukemia (CLL). A pivotal and apical molecule in the BCR pathway is Bruton’s tyrosine kinase (BTK). Together, BTK, Bcl-2, and Mcl-1 participate in the maintenance, migration, proliferation, and survival of CLL cells. Several ongoing and published clinical trials in CLL reported high rates of remission, namely, undetectable measurable residual disease (u-MRD) status with combined BTK inhibitor ibrutinib and Bcl-2 antagonist, venetoclax. While the majority of patients achieve complete remission with undetectable-measurable residual disease, at least one third of patients do not achieve this milestone. We hypothesized that cells persistent during ibrutinib and venetoclax therapy may be sensitive to combined venetoclax and Mcl-1 inhibitor, AMG-176. To test this hypothesis, we took peripheral blood samples at baseline, after Cycle 1 and Cycle 3 of ibrutinib monotherapy, after one week and 1 cycle of ibrutinib plus venetoclax therapy. These serial samples were tested for pharmacodynamic changes and treated in vitro with AMG-176 or in combination with venetoclax. Compared to C1D1 cells, residual cells during ibrutinib and venetoclax treatment were inherently resistant to endogenous cell death. Single agent exposure induced some apoptosis but combination of 100 nM venetoclax and 100 or 300 nM of AMG-176 resulted in 40–100% cell death in baseline samples. Cells obtained after four cycles of ibrutinib and one cycle of venetoclax, when treated with such concentration of venetoclax and AMG-176, showed 10–80% cell death. BCR signaling pathway, measured as autophosphorylation of BTK was inhibited throughout therapy in all post-therapy samples. Among four anti-apoptotic proteins, Mcl-1 and Bfl-1 decreased during therapy in most samples. Proapoptotic proteins decreased during therapy. Collectively, these data provide a rationale to test Mcl-1 antagonists alone or in combination in CLL during treatment with ibrutinib and venetoclax.

Interferon Regulatory Factor 4 (IRF4) Is a Master Regulator in the Pathogenesis of Chronic Lymphocytic Leukemia Patients Harboring Trisomy 12

BACKGROUND:Chronic lymphocytic leukemia (CLL) represents the most common leukemia in the Western countries. Although its natural history is not fully elucidated, it may be determined by a combination of genetic factors and by the complex relationship that leukemic cells entertain with the surrounding microenvironment. Interferon regulatory factor 4 (IRF4, also known as MUM1) is a transcriptional regulator that exerts critical functions in different cell types of the immune system. In B cells IRF4 plays a role in the function and development, by controlling the transition from cycling pre-B to small pre-B cells and by regulating receptor editing, germinal center (GC) reaction and plasma cell differentiation. Deregulation of the biological programs regulated by IRF4 has been related to the pathogenesis of different B-cell malignancies. Tumor cells in CLL patients express reduced levels of IRF4 protein compared to normal B cells and a causal relationship between low levels of IRF4 and the development of CLL has been demonstrated by two elegant in vivo mouse studies.AIM OF THE WORK:The goal of our study was to characterize the expression of IRF4 in CLL patients and to identify its biological functions in order to devise a possible involvement of IRF4 in the pathogenesis of CLL.METHODS:IRF4 expression was monitored by real time PCR in purified CLL cells. Modulation of IRF4 expression in vitro was performed by using a Myc-DDK-tagged IRF4 vector or by siRNA strategy.RESULTS:Among a large cohort of CLL samples (n=250), we identified a peculiar lower expression of IRF4 in patients harboring trisomy 12 (Tri12) compared with other cytogenetic aberrations. We focused our attention on the biological features of Tri12 CLL cells as adhesion, migration and viability dissecting the underlying molecular aspects.First, we tested the ability of Tri12 versus noTri12 CLL cells to adhere to VCAM1 coated plate. As expected, Tri12 CLL cells showed a higher ability to adhere to VCAM1 compared to no Tri12 due to high CD49d expression. We correlated the low expression of IRF4 in Tri12 CLL cells with an increased ability of leukemic cells to adhere to VCAM1 and endothelial cells. Forcing the expression of IRF4 by using a IRF4 vector is able to revert the adhesive features of CLL cells decreasing the surface expression of CD49d. We compared the capacity of Tri12 versus no Tri12 CLL cells to migrate towards CXCL12 in chemotaxis assay. Tri12 CLL cells tended to migrate towards CXCL12 at higher level than no Tri12 CLL cells. The induction of IRF4 in Tri12 CLL cells affected the expression of CXCR4 determining an impaired migration either in response or not to CXCL12. This result is in line with a reduced activation of signaling pathway induced by the reconstitution of IRF4 expression upon CXCL12 stimulation. Investigating the spontaneous apoptotic response of Tri12 CLL cells and no Tri12 CLL cells, we found a prominent resistance to spontaneous death in the first compared to the second subset. Since IRF4 is a regulator of Notch2 during CLL development, we compared the expression of Notch2 and its targets in Tri12 and no Tri12 CLL cells. At the molecular level, Tri12 CLL cells are characterized by an increased expression of Notch2 that is linked to a more pronounced expression of Mcl1 and Hes1 compared to no Tri12 CLL cells. Again, forcing the expression of IRF4 in Tri12 CLL cells, there was a dramatic drop in CLL cells viability that correlates with a reduced expression of Notch2, Mcl1 and CD23. Conversely, reducing the expression of IRF4 by using a siRNA strategy restores Tri12 CLL cells viability by inducing Notch2, Mcl1 and CD23 expression. Finally, we interfered with Notch2 expression using a siRNA or a pharmacological strategy. Silencing of Notch2 in Tri12 CLL cells affected CLL cells viability through the downregulation of CD23 and Mcl1 expression. On this line, using the pharmacological compound gliotoxin, known to interfere with Notch signaling pathway, we found a strong induction of CLL cells apoptosis due to the complete Notch2 degradation.CONCLUSIONS:Collectively, these results indicate that IRF4 plays a relevant role in determining the pathobiology of CLL harboring trisomy 12. The forced restoration of IRF4 expression is followed by marked decrease in adhesion and homing and induction of intrinsic apoptosis in Tri12 CLL cells, suggesting that targeting IRF4 expression may be effective for Tri12 CLL. DisclosuresNo relevant conflicts of interest to declare.

CD150 and CD180 are negative regulators of IL-10 expression and secretion in chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) is a strikingly heterogeneous disease both at the molecular level and clinical disease course. The malignant B cells obtain key proliferation and survival signals within lymph nodes or bone marrow. Moreover, CLL B cells and tumor microenvironment dynamically co-evolve organizing inflammatory and immunosuppressive microenvironment by direct contact with surrounding cells and secretion of cytokines, growth factors, or extracellular vesicles. Finding a way to weaken obtaining by malignant B cells supportive signals may improve CLL outcome and optimize treatment strategies. The aim of this study was to evaluate whether CD150 and CD180 cell surface receptors could be involved in the regulation of CLL B cells-derived cytokines (CCL3, CCL4, IL-6, and IL-10). The study was performed on malignant B cells isolated from peripheral blood of primary CLL patients. Flow cytometry, qPCR, ELISA, western blot, ex vivo cell surface ligation assay, ex vivo drug sensitivity assay, and cell viability assay were used in this study. The CCL3, CCL4, IL-6, and IL-10 mRNA expression levels were heterogeneously presented among studied CLL cases. The elevated CCL3/CCL4 and decreased IL-6/IL-10 expression level are specific features of CLL B cells with positive CD150 and CD180 expression status. Ligation of CD150 and CD180 receptors did not affect CCL3/CCL4 mRNA expression level in CLL B cells, while IL-6 and IL-10 were significantly decreased. After malignant B cells stimulation via CD150 and CD180 observed reduced IL-10 but not IL-6 levels in culture supernatants. Ligation of CD150 and CD180 was linked to the classical NF-κB pathway via regulation of phosphorylation level of NF-κB inhibitor IκBα. We found several correlations between basal mRNA expression levels of CCL3, CCL4, IL-6, and IL-10 in CLL B cells and their sensitivity to chemotherapeutic drugs ex vivo. High CCL3/CCL4 and low IL-10 mRNA expression levels are associated with malignant B cells' sensitivity to BEN, while high IL-6 levels are a sign of CLL B cells' resistance to FC. The revealed involvement of CD150 and CD180 in cytokine regulation expands our knowledge of the role of CD150 and CD180 in the pathobiology of CLL and their contribution to a favorable clinical outcome. Determining the cytokines expression levels together with CD150 and CD180 expression status may help to predict the responsiveness of CLL B cells to chemotherapeutic drugs and optimize personalized chemotherapy scheme.

CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB

CLL Cells with 13q- Are Characterized By High BCL2 Levels and CLL Cells with Trisomy-12 By Increased Activation of STAT3 and NF-κB

Altered patterns of global protein synthesis and translational fidelity in RPS15-mutated chronic lymphocytic leukemia

AbstractGenomic studies have recently identified RPS15 as a new driver gene in aggressive and chemorefractory cases of chronic lymphocytic leukemia (CLL). RPS15 encodes a ribosomal protein whose conserved C-terminal domain extends into the decoding center of the ribosome. We demonstrate that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates. On the other hand, we show that mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner. Quantitative mass spectrometry analyses suggest that RPS15 variants may induce additional alterations in the translational machinery, as well as a metabolic shift at the proteome level in HEK293T and MEC-1 cells. These results indicate that CLL-related RPS15 mutations might act following patterns known for other ribosomal diseases, likely switching from a hypo- to a hyperproliferative phenotype driven by mutated ribosomes. In this scenario, loss of translational fidelity causing altered cell proteostasis can be proposed as a new molecular mechanism involved in CLL pathobiology.

The interplay of CD150 and CD180 receptor pathways contribute to the pathobiology of chronic lymphocytic leukemia B cells by selective inhibition of Akt and MAPK signaling

Cell surface expression of CD150 and CD180 receptors in chronic lymphocytic leukemia (CLL) associates with mutational IGHV status and favourable prognosis. Here we show a direct correlation between cell surface expression and colocalization of these receptors on CLL B cells. In the absence of CD150 and CD180 on the cell surface both receptors were expressed in the cytoplasm. The CD150 receptor was colocalized with markers of the endoplasmic reticulum, the Golgi apparatus and early endosomes. In contrast, CD180 was detected preferentially in early endosomes. Analysis of CD150 isoforms differential expression revealed that regardless of CD150 cell surface expression the mCD150 isoform with two ITSM signaling motifs was a predominant CD150 isoform in CLL B cells. The majority of CLL cases had significantly elevated expression level of the soluble sCD150, moreover CLL B cells secrete this isoform. CD150 or CD180 crosslinking on CLL B cells alone led to activation of Akt, mTORC1, ERK1/2, p38MAPK and JNK1/2 networks. Both CD150 and CD180 target the translation machinery through mTOR independent as well as mTOR dependent pathways. Moreover, both these receptors transmit pro-survival signals via Akt-mediated inhibition of GSK3β and FOXO1/FOXO3a. Unexpectedly, coligation CD150 and CD180 receptors on CLL B cells led to mutual inhibition of the Akt and MAPK pathways. While CD150 and CD180 coligation resulted in reduced phosphorylation of Akt, ERK1/2, c-Jun, RSK, p70S6K, S6RP, and 4E-BP; it led to complete blocking of mTOR and p38MAPK phosphorylation. At the same time coligation of CD150 and CD40 receptors did not result in Akt and MAPK inhibition. This suggests that combination of signals via CD150 and CD180 leads to blocking of pro-survival pathways that may be a restraining factor for neoplastic CLL B cells propagation in more than 50% of CLL cases where these receptors are coexpressed.

Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia.

The crosstalk between chronic lymphocytic leukemia (CLL) cells and tumor microenvironment is essential for leukemic clone maintenance, supporting CLL cells survival, proliferation and protection from drug-induced apoptosis. Over the past years, the role of several soluble factors involved in these processes has been studied. CLL cells express higher levels of endothelin 1 (ET-1) and ETA receptor as compared to normal B cells. Upon ET-1 stimulation, CLL cells improve their survival and proliferation and reduce their sensitivity to the phosphoinositide-3-kinase δ inhibitor idelalisib and to fludarabine. Here, we demonstrate that CLL cells express not only ETA receptor but also ETB receptor. ET-1 acts as a homing factor supporting CLL cells migration and adhesion to microenvironmental cells. In addition, ET-1 stimulates a pro-angiogenic profile of CLL cells increasing VEGF expression through hypoxia-inducible factor-1 (HIF-1α) accumulation in CLL cells. Macitentan, a specific dual inhibitor of ETA and ETB receptors, targets CLL cells affecting leukemic cells migration and adhesion and overcoming the pro-survival and proliferation signals mediated by microenvironment. Furthermore, macitentan cooperates with ibrutinib inhibiting the BCR pathway and with ABT-199 disrupting BCL2 pathway. Our data describe the biological effects of a new drug, macitentan, able to counteract essential processes in CLL pathobiology as survival, migration, trafficking and drug resistance. These findings envision the possibility to interfere with ET receptors activity using macitentan as a possible novel therapeutic strategy for CLL patients.

Constitutive Phosphorylation of STAT3 by the CK2-BLNK-CD5 Complex.

In chronic lymphocytic leukemia (CLL), STAT3 is constitutively phosphorylated on serine 727 and plays a role in the pathobiology of CLL. However, what induces constitutive phosphorylation of STAT3 is currently unknown. Mass spectrometry was used to identify casein kinase 2 (CK2), a serine/threonine kinase that coimmunoprecipitated with serine phosphorylated STAT3 (pSTAT3). Furthermore, activated CK2 incubated with recombinant STAT3 induced phosphorylation of STAT3 on serine 727. Although STAT3 and CK2 are present in normal B- and T cells, STAT3 is not constitutively phosphorylated in these cells. Further study found that CD5 and BLNK coexpressed in CLL, but not in normal B- or T cells, are required for STAT3 phosphorylation. To elucidate the relationship of CD5 and BLNK to CK2 and STAT3, STAT3 was immunoprecipitated from CLL cells, and CK2, CD5, and BLNK were detected in the immunoprecipitate. Conversely, STAT3, CD5, and BLNK were in the immunoprecipitate of CLL cells immunoprecipitated with CK2 antibodies. Furthermore, siRNA knockdown of CD5 or BLNK, or treatment with CD5-neutralizing antibodies significantly reduced the levels of serine pSTAT3 in CLL cells. Finally, confocal microscopy determined that CD5 is cell membrane bound, and fractionation studies revealed that the CK2/CD5/BLNK/STAT3 complex remains in the cytoplasm, whereas serine pSTAT3 is shuttled to the nucleus.Implications: These data show that the cellular proteins CK2, CD5, and BLNK are required for constitutive phosphorylation of STAT3 in CLL. Whether this protein complex phosphorylates other proteins or inhibiting its activity would have clinical benefit in patients has yet to be determined. Mol Cancer Res; 15(5); 610-8. ©2017 AACR.

Whole-exome sequencing in relapsing chronic lymphocytic leukemia: clinical impact of recurrent RPS15 mutations

AbstractFludarabine, cyclophosphamide, and rituximab (FCR) is first-line treatment of medically fit chronic lymphocytic leukemia (CLL) patients; however, despite good response rates, many patients eventually relapse. Although recent high-throughput studies have identified novel recurrent genetic lesions in adverse prognostic CLL, the mechanisms leading to relapse after FCR therapy are not completely understood. To gain insight into this issue, we performed whole-exome sequencing of sequential samples from 41 CLL patients who were uniformly treated with FCR but relapsed after a median of 2 years. In addition to mutations with known adverse-prognostic impact (TP53, NOTCH1, ATM, SF3B1, NFKBIE, and BIRC3), a large proportion of cases (19.5%) harbored mutations in RPS15, a gene encoding a component of the 40S ribosomal subunit. Extended screening, totaling 1119 patients, supported a role for RPS15 mutations in aggressive CLL, with one-third of RPS15-mutant cases also carrying TP53 aberrations. In most cases, selection of dominant, relapse-specific subclones was observed over time. However, RPS15 mutations were clonal before treatment and remained stable at relapse. Notably, all RPS15 mutations represented somatic missense variants and resided within a 7 amino-acid, evolutionarily conserved region. We confirmed the recently postulated direct interaction between RPS15 and MDM2/MDMX and transient expression of mutant RPS15 revealed defective regulation of endogenous p53 compared with wild-type RPS15. In summary, we provide novel insights into the heterogeneous genetic landscape of CLL relapsing after FCR treatment and highlight a novel mechanism underlying clinical aggressiveness involving a mutated ribosomal protein, potentially representing an early genetic lesion in CLL pathobiology.

The pan phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) blocks survival, adhesion and proliferation of primary chronic lymphocytic leukemia cells.

The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia.

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

Axl Regulates Fibroblast Growth Factor Receptor Signaling in B-Cell Chronic Lymphocytic Leukemia: Dual Targeting in CLL Therapy

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease and represents a significant health problem in the western world. We and others have reported that primary CLL B-cells spontaneously produce increased levels of proangiogenic basic fibroblast growth factor (bFGF) in vitro and that most CLL plasma contains elevated levels of bFGF. However, the precise role of bFGF in CLL pathobiology is not clearly understood. In this study we investigated the functional implication of the FGF/FGF receptor (FGFR) signaling axis in CLL B-cell biology.We have detected expression of FGFR1 and FGFR3 with comparatively higher levels of the latter receptor tyrosine kinase (RTK), but no or notably low levels of FGFR2/FGFR4, by flow cytometry and Western blot analyses in primary CLL B-cells. This observation was further supported by detection of FGFR1/FGFR3 transcripts in CLL B-cells by semi-quantitative reverse transcriptase polymerase chain reaction. Although both FGFR1 and FGFR3 in CLL B-cells remain as constitutively phosphorylated, we found significantly higher levels of phosphorylation on FGFR3 and thus this latter receptor is likely the predominant RTK of the FGFR family in these leukemic B-cells. Of note, in vitro stimulation of FGFRs with recombinant bFGF was unable to increase total phosphorylation on FGFRs from their constitutive basal levels in CLL B-cells. Further analysis using a bFGF neutralizing antibody suggested that FGFR phosphorylation in CLL B-cells is likely independent of bFGF ligation. We then interrogated the mechanism of how FGFRs were being phosphorylated and/or maintained at the observed constitutive levels of phosphorylation in CLL B-cells.Our previous studies established that Axl is a critical RTK in CLL B-cells since it acts as a docking site for multiple cellular kinases/lipase, an observation supported by earlier literatures in human malignancies. Given this, Axl is likely capable of cross talk with other RTKs including FGFRs to regulate FGFR-signaling in CLL B-cells. Therefore, in an effort to determine whether Axl is functionally associated with FGFR, we examined if these two RTKs exist in the same molecular complex in CLL B-cells. Indeed, immunoprecipitation assays demonstrated that Axl formed a complex with FGFR3 in CLL B-cells, suggesting that Axl is likely functionally linked to the FGFR signaling. In this regard we found that Axl inhibition, using a high-affinity Axl inhibitor (TP-0903; Tolero Pharmaceuticals), resulted in significant reduction of total FGFR phosphorylation in CLL B-cells. Additionally, siRNA-mediated partial depletion of Axl in CLL B-cells reduced total FGFR phosphorylation. In contrast, inhibition of FGFR phosphorylation using a high-affinity FGFR inhibitor could not alter phosphorylation levels on Axl RTK in CLL B-cells. Together, these findings suggest that Axl has a dominant role in the regulation of FGFR signaling in CLL B-cells.To find out if inhibition of FGFR can induce apoptosis in CLL B-cells we used a specific inhibitor for FGFR (TKI-258; Novartis) to treat CLL B-cells. Here we found a substantial level of apoptosis induction in the leukemic B-cells with a mean LD50 dose of ~2.5 μM. Interestingly, Axl inhibition by TP-0903 induced a robust level of apoptosis in CLL B-cells in the nanomolar dose range with a mean LD50 dose of 0.14 mM. Thus Axl inhibition exerts a very robust cytotoxic effect on CLL B-cell survival likely targeting both Axl and FGFR signaling pathways via Axl inhibition.In conclusion, we have detected expression of constitutively active FGFR1 and 3 in primary CLL B-cells and that inhibition of FGFR signaling induces considerable levels of CLL B-cell apoptosis albeit lower than that observed on Axl RTK inhibition. Interestingly, our findings here suggest that Axl forms an active RTK complex with FGFR and that Axl inhibition modifies FGFR phosphorylation levels. Thus it is likely that Axl RTK can regulate FGFR signaling in the CLL B-cells. In total these observations suggest that the finding of robust induction of apoptosis in CLL B-cells is as a result of targeting two signaling pathways with Axl inhibition: Axl and FGFR. These studies further support investigation of Axl inhibition as a way to develop a more effective and efficient therapeutic intervention for CLL patients. DisclosuresWarner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Kay:Genetech: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; Celgene: Membership on an entity’s Board of Directors or advisory committees; Gilead: Membership on an entity’s Board of Directors or advisory committees.

Endothelin-1 promotes survival and chemoresistance in chronic lymphocytic leukemia B cells through ETA receptor.

The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ETAR and ETBR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ETAR axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ETA receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ETAR triggering. Moreover, the blockade of ETAR by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ETAR via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ETAR blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ETAR signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ETAR inhibition.

Variable induction of PRDM1 and differentiation in chronic lymphocytic leukemia is associated with anergy

AbstractDespite antigen engagement and intact B-cell–receptor (BCR) signaling, chronic lymphocytic leukemia (CLL) cells fail to undergo terminal differentiation. We hypothesized that such failure may be due to anergy, as CLL cells exhibit variable levels of nonresponsiveness to surface IgM stimulation that is reversible in vitro. Moreover, anergy is associated with reduced differentiation capacity in normal B cells. We investigated responses of CLL cells to two potent differentiation-promoting agents, IL-21 and cytosine guanine dinucleotide-enriched oligo-deoxynucleotides. The induction of PR domain-containing protein 1 (PRDM1; also known as Blimp-1), a critical regulator of plasmacytic differentiation, by these agents was closely correlated but varied between individual cases, despite functionally intact IL-21 receptor– and Toll-like receptor 9–mediated signal transducer and activator of transcription 3, and nuclear factor-κB pathways. PRDM1 induction was inversely correlated with the extent of anergy as measured by the ability to mobilize intracellular Ca2+ following BCR crosslinking. PRDM1 responsiveness was associated with other markers of differentiation and proliferation but not with differences in apoptosis. The ability to induce PRDM1 did correlate with differential transcriptional and epigenetic regulation of the PRDM1 gene. These studies extend our understanding of CLL pathobiology, demonstrating that reduced differentiation capacity may be a consequence of anergy. Epigenetic drugs may offer possibilities to reactivate PRDM1 expression as part of novel differentiation therapy approaches.

Evolving Understanding of the CLL Genome

Over the past few years, massively parallel sequencing technologies have revealed with high resolution the tremendous genetic and epigenetic heterogeneity in chronic lymphocytic leukemia (CLL). We have learned how the molecular architecture differs not only between affected individuals but also within samples and over time. These insights have catalyzed our understanding of the pathobiology of CLL and point to critical signaling pathways in the development and progression of the disease. Several key driver alterations have been identified, which serve to refine prognostic schemata but also to inspire the development of new therapeutic strategies. Ongoing advances in technology promise to further elucidate the molecular basis of CLL, and this knowledge is anticipated to aid us in understanding and addressing the clinical challenge presented by the vast variability in the clinical course of patients with CLL.