Signaling In Chronic Lymphocytic Leukemia Research Articles

IL-1 receptor antagonism reveals a yin-yang relationship between NFκB and interferon signaling in chronic lymphocytic leukemia

Nuclear factor kappa B (NFκB) is a pathogenic factor in chronic lymphocytic leukemia (CLL) that is not addressed specifically by current therapies. NFκB is activated by inflammatory factors that stimulate toll-like receptors (TLRs) and receptors for interleukin-1 (IL-1) family members. IL-1 is considered a master regulator of inflammation, and IL-1 receptor signaling is inhibited by the IL-1 receptor antagonist anakinra. These considerations suggested that anakinra might have a role in the treatment of CLL. Consistent with this idea, anakinra inhibited spontaneous and TLR7-mediated activation of the canonical NFκB pathway in CLL cells in vitro. However, CLL cells exhibited only weak signaling responses to IL-1 itself, and anakinra was found to inhibit NFκB along with oxidative stress in an IL-1 receptor-independent manner. Anakinra was then administered with minimal toxicity to 11 previously untreated CLL patients in a phase I dose-escalation trial (NCT04691765). A stereotyped clinical response was observed in all patients. Anakinra lowered blood lymphocytes and lymph node sizes within the first month that were associated with downregulation of NFκB and oxidative stress in the leukemia cells. However, inhibition of NFκB was accompanied by upregulation of type 1 interferon (IFN) signaling, c-MYC-regulated genes and proteins, and loss of the initial clinical response. Anakinra increased IFN signaling and survival of CLL cells in vitro that were, respectively, phenocopied by mitochondrial antioxidants and reversed by IFN receptor blocking antibodies. These observations suggest that anakinra has activity in CLL and may be a useful adjunct for conventional therapies as long as compensatory IFN signaling is blocked at the same time.

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

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

Phosphoproteomics Reveals the Role of Constitutive KAP1 Phosphorylation by B-cell Receptor Signaling in Chronic Lymphocytic Leukemia.

This phosphoproteomic analysis and functional validation illuminated the phosphorylation of KAP1 at S473 as an important downstream BCR signaling event and a potential indicator for the success of ibrutinib treatment in CLL.

MiR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

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.

Proliferative Signals in Chronic Lymphocytic Leukemia; What Are We Missing?

Chronic lymphocytic leukemia (CLL) cells cycle between lymphoid tissue sites where they actively proliferate, and the peripheral blood (PB) where they become quiescent. Strong evidence exists for a crucial role of B cell receptor (BCR) triggering, either by (self-)antigen or by receptor auto-engagement in the lymph node (LN) to drive CLL proliferation and provide adhesion. The clinical success of Bruton’s tyrosine kinase (BTK) inhibitors is widely accepted to be based on blockade of the BCR signal. Additional signals in the LN that support CLL survival derive from surrounding cells, such as CD40L-presenting T helper cells, myeloid and stromal cells. It is not quite clear if and to what extent these non-BCR signals contribute to proliferation in situ. In vitro BCR triggering, in contrast, leads to low-level activation and does not result in cell division. Various combinations of non-BCR signals delivered via co-stimulatory receptors, Toll-like receptors (TLRs), and/or soluble cytokines are applied, leading to comparatively modest and short-lived CLL proliferation in vitro. Thus, an unresolved gap exists between the condition in the patient as we now understand it and applicable knowledge that can be harnessed in the laboratory for future therapeutic applications. Even in this era of targeted drugs, CLL remains largely incurable with frequent relapses and emergence of resistance. Therefore, we require better insight into all aspects of CLL growth and potential rewiring of signaling pathways. We aim here to provide an overview of in vivo versus in vitro signals involved in CLL proliferation, point out areas of missing knowledge and suggest future directions for research.

SLAMF receptors negatively regulate B cell receptor signaling in chronic lymphocytic leukemia via recruitment of prohibitin-2

We identified a subset of Chronic Lymphocytic Leukemia (CLL) patients with high Signaling Lymphocytic Activation Molecule Family (SLAMF) receptor-related signaling that showed an indolent clinical course. Since SLAMF receptors play a role in NK cell biology, we reasoned that these receptors may impact NK cell-mediated CLL immunity. Indeed, our experiments showed significantly decreased degranulation capacity of primary NK cells from CLL patients expressing low levels of SLAMF1 and SLAMF7. Since the SLAMFlow signature was strongly associated with an unmutated CLL immunoglobulin heavy chain (IGHV) status in large datasets, we investigated the impact of SLAMF1 and SLAMF7 on the B cell receptor (BCR) signaling axis. Overexpression of SLAMF1 or SLAMF7 in IGHV mutated CLL cell models resulted in reduced proliferation and impaired responses to BCR ligation, whereas the knockout of both receptors showed opposing effects and increased sensitivity toward inhibition of components of the BCR pathway. Detailed molecular analyzes showed that SLAMF1 and SLAMF7 receptors mediate their BCR pathway antagonistic effects via recruitment of prohibitin-2 (PHB2) thereby impairing its role in signal transduction downstream the IGHV-mutant IgM-BCR. Together, our data indicate that SLAMF receptors are important modulators of the BCR signaling axis and may improve immune control in CLL by interference with NK cells.

Cross-Talk between Cytokine and NF-ĸb Signaling in the CLL Microenvironment Can Affect Sensitivity for Venetoclax

INTRODUCTION. The Bcl-2 inhibitor Venetoclax provides profound reductions in circulating chronic lymphocytic leukemia (CLL) cells in the majority of patients. However, lymph node (LN) responses are less robust, which may be linked to an acquired resistance imposed by pro-survival signals. Prime among these is CD40 stimulation leading to activation of NF-kB, and induction of Bcl-XL expression1. Bcl-XL is a prime determinant of resistance to Venetoclax2 and regulatory mechanisms of its expression are of clinical significance. Cytokines IL-21 and IL-4 are secreted by T helper cells and abundant in the CLL lymph node microenvironment. Importantly, IL-21 and IL-4 play an important role in CLL survival and proliferation3. In the present study, we investigated how signals from T helper cytokines IL-21 or IL-4 affect Bcl-XL expression as a model for the CLL LN microenvironment, specifically in relation to Venetoclax resistance. RESULTS. Following CD40 stimulation, IL-21 and IL-4 show opposing effects on Bcl-XL expression. Correspondingly, this was associated with CD40-induced resistance to Venetoclax which was augmented by IL-4 and reversed by IL-21. We subsequently investigated the rewiring between CD40 activation, differential cytokine signaling and Bcl-XL expression. IL-21 or IL-4 stimulation correspond with differential STAT3 or -6 phosphorylation and STAT3 and -6 have predicted binding sites near the known p65 and p52 binding sites in the Bcl-XL promoter region. Using reporter assays with Bcl-XL promotor constructs we demonstrate competition (through IL-21-induced STAT3) or synergy (through IL-4 induced-STAT6) with CD40-mediated activation of the NF-kB pathway. By applying in situ proximity ligation (isPLA) in primary CLL cells, we showed direct interaction of both (non-)canonical p65 and p52 with STAT3 and STAT6. Moreover, time-course analyses indicated that STAT3 drives NF-kB out of the nucleus, whereas STAT6 keeps NF-kB inside the nucleus, and this distinction controls Bcl-XL expression. These observations suggest that cross-talk between JAK/STAT signaling and NF-kB signaling happens by direct binding to the Bcl-XL promoter and by limiting NF-kB availability at the Bcl-XL promoter. CONCLUSIONS. These data show that protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with JAK/STAT and NF-kB signaling, providing novel therapeutic clues in case of emerging resistance to targeted drugs such as Venetoclax. 1 J. Tromp, S. Tonino, J. Elias, A. Jaspers, D. Luijks, A. Kater, R. van Lier, M. van Oers, E. Eldering. Dichotomy in NF-kB signaling and chemoresistance in IGHV mutated versus unmutated CLL cells upon CD40/TLR9 triggering. Oncogene 2010. 2 R. Thijssen, E. Slinger, K. Weller, C. Geest, T. Beaumont, M. van Oers, A. Kater, E. Eldering. Resistance to ABT-199 induced by microenvironmental signals in chronic lymphocytic leukemia can be counteracted by CD20 antibodies or kinase inhibitors. Haematologica 2015. 3 C. Schleiss, W. Ilias, O. Tahar, Y. Güler, L. Miguet, C. Mayeur-Rousse, L. Mauvieux, L. Fornecker, E. Toussaint, R. Herbrecht, F. Bertrand. M. Maumy-Bertrand, T. Martin, S. Fournel, P. Georgel, S. Bahram, L. Vallat. BCR-associated factors driving chronic lymphocytic leukemia cells in proliferation ex vivo. Scientific Reports 2019. Disclosures Eldering: Celgene: Research Funding; Roche: Research Funding; Janssen Pharmaceutical Companies: Research Funding.

Constitutive Activation of the B Cell Receptor Underlies Dysfunctional Signaling in Chronic Lymphocytic Leukemia

SUMMARYIn cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features—bimodality, hypersensitivity, and hysteresis—in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling.

Constitutive Activation of BCR Explains Dysfunctional Signaling in Chronic Lymphocytic Leukemia

In cancer biology, functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For Chronic Lymphocytic Leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B-cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B-cells toward malignancy. We reveal emergent dynamic features, namely bimodality, hypersensitivity, and hysteresis, in the BCR signaling pathway of primary CLL B-cells. Such signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of signaling motifs, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a novel quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel to genomic profiling.

KRAS, NRAS and BRAF Mutations Are Highly Enriched in TRI12 Chronic Lymphocytic Leukemia and Are Associated to Shorter Time to First Treatment

BackgroundExtensive sequencing data highlighted the recurrence of mutations resulting in constitutive MAP kinase signaling in chronic lymphocytic leukemia (CLL), and proposed these mutations as late drivers of CLL progression (X.S. Puente, Nature, 2015; D.A. Landau, Nature, 2015). Although some preliminary studies suggested KRAS, NRAS and BRAF mutations as possibly associated with trisomy 12 (TRI12) and/or unmutated (UM) IGHV genes (C.D. Herling, Blood, 2016; K. Takahashi, Blood, 2018), dedicated analysis of these aspects is still missing.AimTo correlate the occurrence of KRAS, NRAS and BRAF mutations with specific clinico-biological features in CLL.MethodsThe study included 534 primary CLL purposely enriched with 300 TRI12 cases: 190 cases with TRI12 as sole chromosomal aberration (TRI12only) and 110 cases harboring TRI12 plus other chromosomal aberrations (TRI12plus). The cohort was also enriched in cases with NOTCH1 aberrations (n=214) to evaluate their possible role in RAS-RAF mutations incidence.Amplicon-based targeted next-generation sequencing assay was performed using Miseq Illumina on DNA from purified CLL samples. Analyzed amplicons mapped in the hotspot regions of KRAS, NRAS (exons 2, 3 and 4) and BRAF (exons 11 and 15) genes. Variant allele frequency (VAF) was obtained by Miseq report. CLL cases were characterized for IGHV mutational status, main cytogenetic abnormalities, NOTCH1 and TP53 aberrations. Time to first treatment (TTFT) data were correlated with molecular findings.ResultsWe found 90 missense point mutations (Fig.1) in 64 CLL cases, with prevalence of KRAS (44 mutations in 38 patients), followed by BRAF (31 mutations in 24 patients) and NRAS (15 mutations in 13 patients). Nearly all mutations have been previously associated with gain-of-function phenotype and increased RAS/ERK downstream signaling. The co-occurrence of 2 mutated genes (mainly KRAS and BRAF) were observed in 11 patients. Mutations were mainly subclonal (median VAF 6.15%, range 1.3%-61.6%) with only one third of mutations (27/90) above 15% VAF.A high association between the presence of KRAS/NRAS/BRAF mutations and UM IGHV and the presence of TRI12 was observed. Overall, 87.3% of KRAS/NRAS/BRAF mutated cases had a UM IGHV (p<0.0001) and 79.7% were TRI12 CLL (p<0.0001) (Fig.2). Moreover, in the context of the UM IGHV CLL group (332 cases), TRI12 CLL had a 4-fold higher KRAS/NRAS/BRAF mutation frequency (46/182, 25.3%) than non-TRI12 (9/150, 6%; p<0.0001). Indeed, we found 29/182 (15.9%) KRAS, 9/182 (4.9%) NRAS and 17/182 (9.3%) BRAF mutated cases in the TRI12 group vs. 4/150 (2.7%) KRAS, 3/150 (2%) NRAS and 3/150 (2%) BRAF mutated cases in the non-TRI12 group. The KRAS/NRAS/BRAF mutation frequency was even higher when considering the TRI12only group in the context of UM IGHV (38/133, 28.6%). Conversely, CLL cases with del(13q) as sole chromosomal aberration (n=94) showed the lowest frequency of KRAS/NRAS/BRAF mutations (2/94, 2.1% in the whole cohort and 2/53, 3.8% in the context of UM IGHV cases).Furthermore, a higher prevalence of KRAS/NRAS/BRAF mutations was found in NOTCH1 wild type (wt) (22.1%) when compared to NOTCH1 mutated (11.2%) cases, in the context of the UM IGHV group (p=0.008), pointing to a mutual exclusivity of these mutations in the pathogenesis of disease.No other significant associations with clinical variables as RAI stage at diagnosis, presence of TP53 mutations or gender were observed.We finally evaluated if KRAS/NRAS/BRAF mutations had an impact on TTFT in the context of UM IGHV/TRI12only/NOTCH1wt, that showed the highest incidence of mutations (26/70, 37%). Both KRAS mutations alone (p=0.005) and KRAS/NRAS mutations (p=0.04) were associated with shorter TTFT (Fig.3).ConclusionsTaken together our data show that KRAS, NRAS and BRAF mutations are almost exclusively found in UM IGHV/TRI12/NOTCH1wt CLL and, in the context of this CLL group the presence of KRAS and NRAS mutations is associated with unfavorable prognosis.The high incidence of KRAS, NRAS and BRAF activating mutations in TRI12 CLL together with the observation of higher level of ERK phosphorylation (S. Decker, Blood, 2012) and higher sensitivity to MEK/ERK inhibitors (S. Dietrich, J Clin Invest, 2018) described in the TRI12, reinforce the evidence of an essential role for MEK/ERK signaling in TRI12 CLL, pointing to this pathway as the most appropriate therapeutic target. [Display omitted] DisclosuresZaja:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria; Celgene: Honoraria, Research Funding; Amgen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria.

In vitro and in vivo evidence for uncoupling of B-cell receptor internalization and signaling in chronic lymphocytic leukemia.

B-cell receptor activation, occurring within lymph nodes, plays a key role in the pathogenesis of chronic lymphocytic leukemia and is linked to prognosis. As well as activation of downstream signaling, receptor ligation triggers internalization, transit to acidified endosomes and degradation of ligand-receptor complexes. Herein, we investigated the relationship between these two processes in normal and leukemic B cells. We found that leukemic B cells, particularly anergic cases lacking the capacity to initiate downstream signaling, internalize and accumulate ligand in acidified endosomes more efficiently than normal B cells. Furthermore, ligation of either surface CD79B, a B-cell receptor component required for downstream signaling, or surface Immunoglobulin M (IgM) by cognate agonistic antibody, showed that the two molecules internalize independently of each other in leukemic but not normal B cells. Since association with surface CD79B is required for surface retention of IgM, this suggests that uncoupling of B-cell receptor internalization from signaling may be due to the dissociation of these two molecules in leukemic cells. A comparison of lymph node with peripheral blood cells from chronic lymphocytic leukemia patients showed that, despite recent B-cell receptor activation, lymph node B cells expressed higher levels of surface IgM. This surprising finding suggests that the B-cell receptors of lymph node- and peripheral blood-derived leukemic cells might be functionally distinct. Finally, long-term therapy with the Bruton’s tyrosine kinase inhibitors ibrutinib or acalabrutinib resulted in a switch to an anergic pattern of B-cell receptor function with reduced signaling capacity, surface IgM expression and more efficient internalization.

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.

SYK inhibition thwarts the BAFF - B-cell receptor crosstalk and thereby antagonizes Mcl-1 in chronic lymphocytic leukemia.

Although small molecule inhibitors of B-cell receptor-associated kinases have revolutionized therapy in chronic lymphocytic leukemia (CLL), responses are incomplete. Pro-survival signaling emanating from the microenvironment may foster therapeutic resistance of the malignant B cells resident in the protective lymphoid niches. B-cell activating factor (BAFF) is critical to the survival of both healthy and neoplastic B cells. However, the pro-survival pathways triggered by BAFF have not been fully characterized. Here we show that BAFF elicited resistance to spontaneous and drug-induced apoptosis in stromal co-cultures, induced activation of both canonical and non-canonical NFκB signaling pathways, and triggered B-cell receptor signaling in CLL cells, independently of IGHV mutational status. SYK, a proximal kinase in the B-cell receptor signaling cascade, acted via STAT3 to bolster transcription of the anti-apoptotic protein Mcl-1, thereby contributing to apoptosis resistance in BAFF-stimulated cells. SYK inhibitor entospletinib downregulated Mcl-1, abrogating BAFF-mediated cell survival. BAFF-B-cell receptor crosstalk in neoplastic B cells was mediated by SYK interaction with TRAF2/TRAF3 complex. Thus, SYK inhibition is a promising therapeutic strategy uniquely poised to antagonize crosstalk between BAFF and B-cell receptor, thereby disrupting the pro-survival microenvironment signaling in chronic lymphocytic leukemia.

Targeting B Cell Signaling in Chronic Lymphocytic Leukemia.

In recent years, a revolution in the management of chronic lymphocytic leukemia (CLL) has centered on the targeting of the B cell receptor (BCR) signaling pathway. Our improved understanding of the biology of cell signaling in CLL and the development of oral kinase inhibitors directed at the BCR pathway has led to the approval of two new agents and has the potential to radically change the treatment of CLL in both the relapsed/refractory and upfront settings. In this review, we will describe the underlying biology of the BCR signaling pathway. We will discuss the landmark clinical trials resulting in the approval of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib. We will highlight ongoing trials that are evaluating the use of combinations of these agents with standard chemotherapy. We will evaluate some of the emerging data regarding toxicity, potential off-target effects, and mechanisms of resistance to BCR signaling pathway blockade. Finally, we will highlight some of the next-generation BCR pathway inhibitors currently in development.

Functional Differences between IgM and IgD Signaling in Chronic Lymphocytic Leukemia.

BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.

Bruton's tyrosine kinase inhibitor restrains Wntsignaling in chronic lymphocytic leukemia.

The B-cell receptor (BCR) signaling pathway serves an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), and has been identified as a novel and effective therapeutic target of CLL, with particular focus its kinase factor, BTK. Previous studies have focused on combining the BTK inhibitor with additional chemotherapeutic agents to improve the prognosis of patients with CLL. Further investigation into the mechanism of the BTK inhibitor would promote an understanding of the pathogenesis of CLL. The current study investigated the association between ibrutinib and the Wnt signaling pathway, additionally focussing upon one of its regulators, metadherin (MTDH), which has been identified to be overexpressed in CLL and is considered a promoter of the Wnt pathway. The experiments in the current study were performed in the MEC-1 CLL cell line. Results indicated that MTDH, β-catenin and lymphoid-enhancing factor-1 were inhibited subsequent to ibrutinib treatment. The results indicate that in CLL, ibrutinib is likely to possess an inhibitory role in Wnt signaling.

Abstract B039: In silico models for B cell receptor signaling in chronic lymphocytic leukemia

Abstract Recent discoveries of ZAP-70 (ζ chain–associated protein kinase of 70 kD) and Syk (spleen tyrosine kinase) inhibitors (gefitinib1 and fostamatinib2 respectively) provide promising therapeutic options for patients with chronic lymphocytic leukemia (CLL). However, the precise action of these inhibitors in the B cell receptor (BCR) signaling pathway is not well understood. This study designs an integrated mechanistic description of the BCR signaling pathway in CLL cells, and investigates the effects of possible inhibitors on this pathway. In this work, we examined several computational models of ZAP-70 and Syk regulation in the BCR signaling pathway to explain the observed differences in the clinical behaviors of ZAP+ / ZAP- phenotypes of B-CLL patients. Specifically, we characterized the effects of different ZAP-70 and Syk expression and phosphorylation levels on the BCR activation threshold. The correlations between the observed and calculated trends are reproduced quantitatively. Our calculations show that depending on the amount of Syk that is expressed in cells, increased ZAP-70 expression is correlated with decreased levels of phosphorylated ZAP-70 and Syk, and vice versa. ZAP-70 and Syk phosphorylation occur independently, but is dependent on the amount of ZAP-70 and Syk that is expressed in cells. These results support reported experimental observations.3 We also find that ZAP-70 is able to compensate for a missing Syk functionality with an increased BCR activation threshold in Syk-deficient B-CLL cells, similar to Syk-deficient B cell study.4 In addition, we find that selective inhibition of either Syk or ZAP-70 will result in a disease relapse. This suggests the used of two inhibitors rather than one for the treatment of ZAP-70+ patients, based on our model. Lastly, we find that enhanced or reduced BCR signaling is observed at low amounts of Syk expression in CLL cells. However, this computational prediction needs to be verified experimentally in order to identify a viable physiological range of the amount of Syk that is expressed in B-CLL cells for ZAP-70+ and ZAP-70- phenotypes. Our results uncover molecular mechanisms of ZAP-70 and Syk regulation in the BCR signaling pathway for its future therapeutic use in B-CLL. Our in silico network analysis will help in the development of immunotherapies targeting ZAP-70 and Syk functions to regulate B-CLL responses. Furthermore, our study provides information about responses to certain cancer therapies, and tumor progression.

Mirna-21 Targets Sprouty2 to Activate SYK Mediated BCR and MAPK-Erk Signaling in Chronic Lymphocytic Leukemia

Abstract INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) represents the most common adult leukemia in the western world. Several reports have demonstrated that the constitutively activated MAPK-Erk signaling pathways promote CLL cell proliferation and survival. However, the precise molecular mechanisms that lead to deregulated MAPK signaling in CLL initiation and progression are not fully understood. We have previously reported that Sprouty2 (Spry2) is a negative regulator of BCR and MAPK-Erk signaling in poor prognosis CLL (American Society of Hematology, 56th Annual Meeting, San Francisco, CA, 2014). Here in follow up studies, we set to further determine molecular basis deregulation of Spry2 in patients with poor prognosis CLL. Spry2 is either epigenetically silenced or targeted by Microrna-21 (miR21) in several malignancies. MiR-21 plays a role of oncomir in CLL, which is significantly upregulated in CLL patients with poor prognosis specifically with Del 17p, High 38 and Zap70 expression. Notably, we observed a decrease in Spry2 expression in CLL cells expressing high levels of miR-21. Also, it is already reported that miR21 is regulated by STAT3 transcription factor in CLL patients with poor prognosis in refractory phase. However the precise molecular mechanism by which the miR21 leads to leukemic progression in poor prognosis CLL is not known. Here we demonstrate the molecular mechanism of miR21 to activate Syk mediated BCR and MAPK-Erk signaling by targeting Spry2 in CLL cells from poor prognosis patients. METHODOLOGY: We isolated CLL cells from peripheral blood of newly diagnosed high CD38 poor prognosis patients. For molecular analysis, in addition to primary CLL cells we use Mec-1 CLL cell line. To measure the levels of miR21 we used Taqman assay qPCR from Invitrogen. To decrease miR21 levels we used miR21 inhibitor (Invitrogen) and STAT3 inhibitor (SantaCruz). To overexpress miR21 we used pcDNAmir-21 construct from Addgene. Activation of BCR and MAPK-Erk signaling pathway was measured by levels of p-Erk and p-Syk. Expression of Spry2, Erk and Syk was measured by western blot analysis. RESULTS: To study if miR21 targets Spry2 in CLL cells, we first overexpressed miR21 in Mec-1 cells. We observed a significant decrease in Spry2 expression in Mec-1 cells expressing high levels of miR-21 compared to empty vector control. In our previous studies we observed Spry2 acts as a negative regulator of BCR and MAPK-Erk signaling. Therefore, we next studied the activity of Spry2 targeting signaling pathways in miR21-overexpressing CLL cells. We observed elevated levels of p-Erk and p-Syk in CLL cells overexpressing miR21. For control we used pcDNA-empty vector. Whereas upon miR21 knockdown in CLL cells resulted in stabilization of Spry2 expression and decrease in BCR and MAPK-Erk signaling. Also, targeting miR21 by STAT3 inhibitor leads to induction of spontaneous apoptosis in CLL cells. We next tested the effect of STAT3 inhibitor on primary CLL cells from 13 different patients. We measured the viability of CLL cells using MTT assay, we observed higher efficacy of STAT3 inhibitor over Syk and BRAF inhibitors on CLL cells. CONCLUSION: We conclude that Spry2 is a target of miR21 in CLL cells from poor prognosis patients. MiR21 targets Spry2 to activate Syk mediated BCR and MAPK signaling in CLL patients. Also, STAT3 can be used as therapeutic target for poor prognosis CLL patients with high miR21 expression. Disclosures No relevant conflicts of interest to declare.

Abstract B44: Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton's tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. The PI3K p110δ inhibitor, idelalisib, was granted a breakthrough therapy designation by the FDA in relapsed CLL and the BTK inhibitor, ibrutinib, has received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, IPI-145, which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of IPI-145 in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM IPI-145 for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to IPI-145, displaying about 20% decrease in viability at concentrations greater than 0.5μM, however the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM IPI-145 for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, IPI-145 is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, IPI-145. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, as IPI-145 can continue to inhibit the PI3K/AKT pathway in the setting of a C481S BTK mutation, it provides justification for clinical studies of this agent in this subset of patients. Citation Format: Amy J. Johnson, Shuai Dong, Daphne Guinn, Jason A. Dubovsky, Yiming Zhong, Amy Lehman, Jeff Kutok, Jennifer A. Woyach, John C. Byrd. Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B44.