Primary Chronic Lymphocytic Leukemia Cells Research Articles

S897 VALIDATION OF A NOVEL MECHANISM OF ACTION FOR TREATMENT OF CCR7+ HEMATOLOGICAL MALIGNANCIES WITH FIRST‐IN‐CLASS ANTIBODY CAP‐100

Background:The homeostatic CC‐chemokine receptor 7 (CCR7) controls trafficking of immune cells to the lymph node (LN). Accordingly, CCR7 is highly expressed in hematological malignancies with nodal involvement including chronic lymphocytic leukemia (CLL), several B‐cell non‐Hodgkin lymphomas (NHL) such as mantle cell lymphoma (MCL), and various mature T‐cell neoplasia like T‐cell prolymphocytic leukemia (T‐PLL) and Sézary syndrome (SS). Upon engagement by its ligands, the chemokines CCL19 and CCL21, CCR7 directs tumor cells to LN. In this protective microenvironment, CCR7 contributes to tumor cell survival and proliferation. Indeed, both high CCR7 surface expression levels and high CCR7‐triggered migratory responses correlate with lymphadenopathy, adverse prognostic factors, and shorter patient survival. Therefore, strategies targeting CCR7 could provide a novel therapeutic approach for CCR7+ hematological malignancies.Aims:In this study we aimed to validate CCR7 as a novel target for antibody‐based therapies in CCR7‐expressing blood cancers.Methods:We have generated CAP‐100, the first humanized IgG1 monoclonal antibody (mAb) that specifically binds to human CCR7 and neutralizes ligand‐mediated signaling, and evaluated the antibody in various in‐vitro and in‐vivo preclinical models.Results:CAP‐100 potently inhibited in vitro migration of primary CLL, MCL, T‐PLL and SS tumor cells. Furthermore, in vivo pre‐clinical studies confirmed that CAP‐100 effectively blocked entry of CCR7‐expressing cells to LNs. CAP‐100 also abrogated survival elicited by CCR7 in CLL, and showed potent antibody‐dependent cell‐mediated cytotoxicity (ADCC) against CLL or CCR7+ T‐lymphomas cells. This Fc‐mediated cell killing activity clearly outperformed anti‐CD20 mAb rituximab and anti‐CD52 mAb alemtuzumab, standards of care in B‐cell and T‐cell lymphomas respectively. In all cases, CAP‐100 activity was independent of prognostic markers for high risk disease. Finally, when given as monotherapy in disseminated B‐NHL and CLL xenograft tumors in SCID mice, CAP‐100 inhibited tumor growth and extended survival significantly. In addition, CAP‐100 strongly inhibited the infiltration into as well as the survival of tumor cells in the LN.Summary/Conclusion:Our results demonstrate that CAP‐100, the first‐in‐class anti‐CCR7 mAb, is a potent antagonist with biological activity in several CCR7+ hematological malignancies, including relapsed/refractory disease. Moreover, these results highlight the relevance of the CCR7‐CCL19/CCL21 pathway as a therapeutic target in these diseases. CAP‐100's unique ability to block migration of tumor cells to the LN (where also inhibits tumor cell survival), in combination with its potent cell killing activity, represents a novel and differentiating mechanism of action. This provides the biological rationale for use of CAP‐100, either as monotherapy or in combination with novel agents. Clinical trials in CLL and CCR7‐expressing NHL will be initiated soon.

PS1145 TARGETED ACTIVATION OF THE PROTEIN PHOSPHATASES SHP-1 AND PP2A ABROGATES CONSTITUTIVE ACTIVATION OF SURVIVAL PATHWAYS IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA (LGLL)

Background: Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Aims: Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Methods: TLR stimulation was performed with an agonist mix of Pam3CSK4, HKLM, FSL1 and ODN2006. Cytokine secretion and CLL proliferation were assessed by Luminex system and carboxyfluorescein succinimidyl ester staining, respectively. For in vivo studies, the Eμ-TCL1 adoptive transfer mouse model was used. Results: Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and –unmutated cases. In the Eμ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Summary/Conclusion: Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

PS1144 TARGETING IRAK4 DISRUPTS INFLAMMATORY PATHWAYS AND DELAYS TUMOR DEVELOPMENT IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Interleukin‐1 receptor‐associated kinase 4 (IRAK4) plays a critical role in Toll‐like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease.Aims:Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL.Methods:TLR stimulation was performed with an agonist mix of Pam3CSK4, HKLM, FSL1 and ODN2006. Cytokine secretion and CLL proliferation were assessed by Luminex system and carboxyfluorescein succinimidyl ester staining, respectively. For in vivo studies, the Eμ‐TCL1 adoptive transfer mouse model was used.Results:Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose‐dependent manner. We further observed a decrease in NF‐κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88‐mutated and –unmutated cases. In the Eμ‐TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells.Summary/Conclusion:Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Pharmacologic inhibition of the ubiquitin-activating enzyme induces ER stress and apoptosis in chronic lymphocytic leukemia and ibrutinib-resistant mantle cell lymphoma cells

With the advent of proteasome inhibitors (bortezomib) and pleiotropic pathway modulators which target cereblon E3 ligase (lenalidomide), the ubiquitin-proteasome system has emerged as a tractable target in non-Hodgkin lymphoma and multiple myeloma. Here we report that TAK-243, a small molecule inhibitor of the ubiquitin-activating enzyme (UAE), induced ER stress and the unfolded protein response in primary chronic lymphocytic leukemia cells, facilitating cell death. Moreover, targeting UAE was effective in ibrutinib-resistant mantle cell lymphoma cell lines and primary cells in vitro. Thus, UAE is a promising target in lymphoid malignancies, including ibrutinib-resistant lymphomas, an area of unmet medical need.

Tumor-Specific Reactive Oxygen Species Accelerators Improve Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies.

Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance.

PIM kinase inhibitor, AZD1208, inhibits protein translation and induces autophagy in primary chronic lymphocytic leukemia cells.

The PIM1, PIM2, and PIM3 serine/threonine kinases play a role in the proliferation and survival of cancer cells. Mice lacking these three kinases were viable. Further, in human hematological malignancies, these proteins are overexpressed making them suitable targets. Several small molecule inhibitors against this enzyme were synthesized and tested. AZD1208, an orally available small-molecule drug, inhibits all three PIM kinases at a low nanomolar range. AZD1208 has been tested in clinical trials for patients with solid tumors and hematological malignancies, especially acute myelogenous leukemia. The present study evaluated the efficacy and biological actions of AZD1208 in chronic lymphocytic leukemia (CLL) cells. CLL cells had higher levels of PIM2 protein and mRNAs than did normal lymphocytes from healthy donors. Treatment of CLL lymphocytes with AZD1208 resulted in modest cell death, whereas practically no cytotoxicity was observed in healthy lymphocytes. To determine the mechanism by which AZD1208 inhibits PIM kinase function, we evaluated PIM kinase pathway and downstream substrates. Because peripheral blood CLL cells are replicationally quiescent, we analyzed substrates involved in apoptosis, transcription, and translation but not cell cycle targets. AZD1208 inhibited protein translation by decreasing phosphorylation levels of 4E-binding protein 1 (4E-BP1). AZD1208 induced autophagy in replicationally-quiescent CLL cells, which is consistent with protein translation inhibition. These data suggest that AZD1208 may elicit cytotoxicity in CLL cells through inhibiting translation and autophagy induction.

Activation of NF-κB in B cell receptor signaling through Bruton's tyrosine kinase-dependent phosphorylation of IκB-α.

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.

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

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

AKT/mTORC2 Inhibition Activates FOXO1 Function in CLL Cells Reducing B-Cell Receptor-Mediated Survival.

To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL). Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib. Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT-dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib. Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells.

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101’s action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL

AbstractNOTCH1 is mutated in 10% of chronic lymphocytic leukemia (CLL) patients and is associated with poor outcome. However, NOTCH1 activation is identified in approximately one-half of CLL cases even in the absence of NOTCH1 mutations. Hence, there appear to be additional factors responsible for the impairment of NOTCH1 degradation. E3-ubiquitin ligase F-box and WD40 repeat domain containing-7 (FBXW7), a negative regulator of NOTCH1, is mutated in 2% to 6% of CLL patients. The functional consequences of these mutations in CLL are unknown. We found heterozygous FBXW7 mutations in 36 of 905 (4%) untreated CLL patients. The majority were missense mutations (78%) that mostly affected the WD40 substrate binding domain; 10% of mutations occurred in the first exon of the α-isoform. To identify target proteins of FBXW7 in CLL, we truncated the WD40 domain in CLL cell line HG-3 via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9). Homozygous truncation of FBXW7 resulted in an increase of activated NOTCH1 intracellular domain (NICD) and c-MYC protein levels as well as elevated hypoxia-inducible factor 1-α activity. In silico modeling predicted that novel mutations G423V and W425C in the FBXW7-WD40 domain change the binding of protein substrates. This differential binding was confirmed via coimmunoprecipitation of overexpressed FBXW7 and NOTCH1. In primary CLL cells harboring FBXW7 mutations, activated NICD levels were increased and remained stable upon translation inhibition. FBXW7 mutations coincided with an increase in NOTCH1 target gene expression and explain a proportion of patients characterized by dysregulated NOTCH1 signaling.

Suppression of minichromosome maintenance 7 expression sensitizes chronic lymphocytic leukemia cells to fludarabine

Chronic lymphocytic leukemia (CLL) constitutes the largest percentage of adult leukemia cases in Western countries. Classically, fludarabine (Flu) is an effective drug used as a first-line therapy for CLL; however, Flu resistance limits its clinical effect. Minichromosome maintenance (MCM) complex components 2–7 exert important functions in maintaining genomic stability. Replication stress occurs upon dysregulation of MCM7, which potentiates malignant phenotypes. In this study, primary CLL cells and CLL-derived cell lines displayed elevated MCM7 expression. In CD40-stimulated primary CLL cells, MCM7 inhibition resulted in increased Flu-induced apoptosis and delayed repair of DNA damage. In the MEC-1 and EHEB cell lines, knockdown of MCM7 with lentivirus significantly inhibited cell proliferation and promoted cell cycle arrest at S phase. Moreover, MCM7 silencing sensitized both cell lines to Flu by increasing replication stress. The combination of Flu administration with MCM7 inhibition represents a novel approach to reverse Flu resistance in CLL.

Dual inhibition of MEK1/2 and AKT by binimetinib and MK2206 induces apoptosis of chronic lymphocytic leukemia cells under conditions that mimic the tumor microenvironment

Several key pathways mediate signaling via the B-cell receptor, including the mitogen-activated protein kinase-ERK1/2 pathway. However, inhibition of MEK1/2, a key component of the MAPK-ERK1/2 signaling cascade, results in paradoxical activation of AKT in chronic lymphocytic leukemia (CLL) cells. In the current study we demonstrate synergy between the MEK1/2 inhibitor binimetinib and the AKT inhibitor MK2206, which combined induce apoptosis of primary CLL cells and restrict the cell cycle progression and proliferation of the OSU-CLL cell line. The mechanisms of action of the drug combination involve dual inhibition of MAPK-ERK1/2 and AKT signaling and down-regulation of Mcl-1 expression. Collectively, these data suggest that dual inhibition of MEK1/2 and AKT may represent a therapeutic option for CLL, capable of overcoming the pro-survival effects of the lymph node and bone marrow microenvironments.

Increased autocrine interleukin-6 production is significantly associated with worse clinical outcome in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains incurable with current standard therapy. We have previously reported that an increased expression of interleukin‐6 (IL‐6) receptor CD126 leads to resistance of CLL cells to chemotherapy and worse prognosis for patients with CLL. In this study, we determine whether autocrine IL‐6 production by CLL B cells is associated with poor clinical outcome and explore IL‐6‐mediated survival mechanism in primary CLL cells. Our results demonstrate that higher levels of autocrine IL‐6 are significantly associated with shorter absolute lymphocyte doubling time, patients received treatment, without complete remission, advanced Binet stages, 17p/11q deletion, and shorter time to first time treatment and progression‐free survival. IL‐6 activated both STAT3 and nuclear factor kappa B (NF‐κB) in primary CLL cells. Blocking IL‐6 receptor and JAK2 inhibited IL‐6‐mediated activation of STAT3 and NF‐κB. Our study demonstrates that an increased autocrine IL‐6 production by CLL B‐cells are associated with worse clinical outcome for patients with CLL. IL‐6 promotes CLL cell survival by activating both STAT3 and NF‐κB through diverse signaling cascades. Neutralizing IL‐6 or blocking IL‐6 receptor might contribute overcoming the resistance of CLL cells to chemotherapy. We propose that the measurement of autocrine IL‐6 could be a useful approach to predict clinical outcome.

Expression and Effects of Ligand-activated Estrogen Receptors in Chronic Lymphocytic Leukemia.

Recent epidemiological data indicate that lymphoid tumors may be influenced by estrogens. The effects of estrogens are mediated via nuclear estrogen receptors α (ERα) and β (ERβ). This study investigated the potential functions of ligand-activated ERs in chronic lymphocytic leukemia (CLL). The ER mRNA expression in B lymphocytes isolated from patients with CLL was analyzed by quantitative real-time polymerase chain reaction. To evaluate the effects of ERβ signaling, primary CLL cells and CLL-derived MEC1 cells were treated with selective ERβ agonists. The mRNA expression of ERα, ERβ1 and its splice variant ERβ2 was detected in CLL cells. Selective ERβ agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile induced apoptosis in primary CLL cells and suppressed the growth of CLL-derived MEC1 cells. A suppressive effect of ERβ agonists on the growth of ERβ-expressing CLL cells was found, indicating that ERβ may be considered as a potential therapeutic target in CLL.

The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

The Fully Human Anti-CD47 Antibody SRF231 Has Dual-Mechanism Antitumor Activity Against Chronic Lymphocytic Leukemia (CLL) Cells and Increases the Activity of Both Rituximab and Venetoclax

DUSP1/6 Inhibition Reduces Tumor Cells and Activates Immune Response in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by an expansion of monoclonal, mature B cells that carry auto/polyreactive B cell receptors (BCRs). It is one of the most prevalent B cell malignancies in Western countries. Over the past decade, extensive research has led to significant improvement in understanding its pathogenesis, particularly in regard to the BCR signaling pathway. This has led to the development of novel and promising treatment options, however, none of these approaches are curative and relapses are challenging to treat. Until now allogeneic hematopoietic stem cell transplantation remains the only treatment option for durable remission in poor-risk disease. The therapeutic difficulties result from the heterogeneity, the advanced age of the patients, chemoresistance and relapse of the disease. Here, we test a novel strategy for simultaneously targeting the malignant cells and reverting the CLL-suppressed immune response.Stimulation of the antigen receptors of immune cells readily induces proliferation, differentiation and functional activation. However, strong binding of antigen can also induce negative selection in lymphocytes. CLL B cells constitutively signal through their BCR and thus we set out to investigate whether they rely on the counterbalancing, negative regulation of specific downstream signaling pathways, in particular the mitogen-activated protein kinase (MAPK) pathway. The dual specificity phosphatases 1 and 6 (DUSP1 and DUSP6) dephosphorylate extracellular-signal regulated kinase 1/2 (ERK1/2) and thereby limit ERK1/2 activation. These molecules are frequently downregulated in solid tumors (Khor et al., Int. J. Med.Sci 2013; Okudela et al., Am. J. Pathol. 2009). We therefore set out to analyze the expression levels of DUSP1 and DUSP6 in CLL and found them readily expressed at various levels, comparable to normal B cells. To determine the functional relevance of DUSP1 and DUSP6, we blocked their phosphatase function using the small molecule inhibitor BCI. By treatment with BCI, we induced hyperactivation of the MAPK signaling cascade followed by cell death of the CLL cells. Interestingly, the induction of cell death is specific for CLL cells and does not occur to the same extent in other malignant B cell lymphoma cells or healthy donor-derived B cells. This deleterious effect of BCI was evident in primary patient-derived CLL cells as well as in the CLL-like cell lines MEC-1 and EHEB and in CLL cells derived from the T cell leukemia/lymphoma 1 (TCL1)-driven mouse model. To further investigate the downstream signaling event upon BCI treatment, we conducted a global phosphoproteome analysis. After treatment of primary CLL cells with BCI, the most significant alterations were within the BCR signaling pathway, including hyperphosphorylation of ERK1/2 and followed by a rapid induction of a DNA damage response. These results were validated by immunoblot analysis of human and murine CLL cells and were not detected in BCI-resistant cell lines.Beside the direct effects of BCI on the CLL cells we set out to investigate effects on other immune cells, directly by BCI and secondary via CLL cells treated with BCI. Indeed, we observed changes in immune cell compartment: BCI-treated CLL patient-derived peripheral blood mononuclear cells (PBMCs) resulted in selective enrichment of cytotoxic T cells. Furthermore, BCI treatment of CLL cells fed with Ovalbumin, in co-culture with or without BMDCs and OT-I cytotoxic CD8 T cells (specifically recognizing the SINFEKL peptide), resulted in the induction of immunogenic cell death of CLL cells. This was evidenced by enhanced antigen-specific T cell proliferation and release of the high mobility group box 1 protein (HMGB1). To investigate indirect effects of BCI, we treated CLL cells with BCI at sublethal doses, then washed the cells and co-cultured these primary CLL cells or CLL cell lines with healthy donor-derived PBMCs. The frequencies of CLL-induced myeloid-derived suppressor cells (MDSCs) as well as regulatory T cells (Tregs) were reduced after co-cultivation of PBMCs with BCI-pretreated CLL cells.Taken together, our data indicate that negative feedback inhibition reduces CLL content by induction of immunogenic cell death and activates immune cells to target the CLL-induced dysfunction of the immune system. We therefore propose that inhibition of DUSP1/6 is a promising therapeutic approach for CLL. DisclosuresNo relevant conflicts of interest to declare.

RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells

We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5‘ UTRs, CDS, and 3‘ UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity.To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. DisclosuresHadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

Comparative Analysis on the Anti-Tumor Activity of Venetoclax and Obinutuzumab (VO) Versus Venetoclax and Rituximab (VR) in Primary CLL Cells, Ex Vivo

INTRODUCTION: Treatment of chronic lymphocytic leukemia (CLL) has expanded significantly with the approval of multiple small molecule inhibitors. This is of great significance for patients with adverse cytogenetic features who tend to respond poorly to standard chemo-immunotherapy (CIT). While single agent ibrutinib and venetoclax (V) have shown high rates of overall response, complete remission and minimal residual disease (MRD) eradication rates remain low. This argues for further testing and development of various combination strategies. The MURANO trial compared venetoclax and rituximab (VR) versus bendamustine and rituximab (BR) in patients with relapsed/refractory CLL reporting clear superiority of VR over BR. MRD rates in the bone marrow were reported to be 27.3% for VR versus 1.5% for BR. Given much higher rates of MRD eradication with combination of small molecule inhibitors and monoclonal antibodies (mAb) compared to standard CIT, we performed a comparative investigation into the direct and immune-mediated cytolytic effects of VR versus V + Obinutuzumab (O, type II anti-CD20 mAb) in primary CLL cells and B-lymphoid cell lines.METHODS: CD19+ B-cells were isolated from PBMCs of CLL patients (N=3). For all experiments using primary CLL cells, concentration of VR and VO was 3nM (V) and 10ug /ml (R, O), respectively. For cell lines, VR and VO was used at 5uM (V) and 10ug /ml (R, O), respectively. Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by VR and VO was assessed in Calcein AM labeled CLL cells or cell lines co-cultured with healthy donor PBMCs (E:T ratio, 40:1); complement-dependent cytotoxicity (CDC) was measured using 10% serum from a healthy human donor.RESULTS: We assessed the ability of V+/-O or V+/-R to induce apoptotic cell death in the CD20+ BCWM.1 cell line (Waldenström's macroglobulinemia [WM] phenotype) and the MEC-1 cell line (B-PLL phenotype); with CD20- RPCI-WM1 (WM cells, negative control). Notably, Bcl-2 protein is expressed in all the aforementioned cell lines. We observed that single agent V, O and R induced ~30%, 61% and 13.64% annexin V/PI positivity in BCWM.1 cells, respectively. However, a significant degree of cell death was noted in VO-treated cells (~74%) compared to VR-treated cells (~40%) (p<0.01). Next, we examined for apoptosis in MEC1 cells and noted a similar trend; where the VO combination induced markedly more cell death (~71%) than VR (~57%). Contrastingly, in RPCI-WM1 cells neither single agent O or R could elicit >12% annexin V/PI positivity and where the addition of V increased apoptosis by only 3 - 4%. We also examined the apoptotic potential of VO or VR in tumor cells from low, intermediate and high-risk CLL patients. In low and intermediate-risk CLL cells from low and intermediate-risk patients, V alone induced ~30% cell death, which increased significantly with the addition of O (VO) to between 48 - 52%. Contrastingly, the combination of VR did not induce more than 29 - 32% apoptosis. In CLL cells from high-risk patient, we noted that exposure to single agent V induced ~ 28% cell death and in VO-treated cells, this number increased to 47%. We also examined for ADCC and CDC in the same cell lines and primary CLL cells. Despite considerable variability, single agent O and VO treatment of tumor cells resulted in greater ADCC than VR treatment. By contrast, in single agent R or VR-treated cells, more CDC was observed.CONCLUSION: Our preliminary investigation in VR- and VO-treated cell lines and primary CLL cells suggests the VO combination may be superior to VR in induction of direct tumor cell death. Mechanistic experiments underway will provide further insight and can aid in design of future VO-based clinical studies in CLL. DisclosuresAilawadhi:Pharmacyclics: Research Funding; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy.

The Dual PI3/PIM-Kinase Inhibitor, Ibl-202, Is Highly Synergistic with Venetoclax Against CLL Cells, and TP53-Knock-out Cells, and Under Conditions That Mimic the Tumor Microenvironment

BackgroundThe B-cell receptor (BCR) signaling pathway and the pro-survival Bcl-2-family of proteins play crucial roles in the pathogenesis of chronic lymphocytic leukemia (CLL). Constitutive activity of the BCR signaling pathway and overexpression of Bcl-2 promote CLL-cell survival, proliferation and drug resistance. BCR-targeted therapies, most notably ibrutinib and idelalisib and the Bcl-2 inhibitor Venetoclax, demonstrate the potential of targeting these pathways. However, there is no evidence that these novel agents are curative in the event of relapse. Treatment options remain limited for patients treated with these agents, in particular those with TP53 lesions.In our recent study (Crassini et al., BJH 2018) we demonstrated efficacy of the PI3/PIM kinase inhibitor, IBL-202 (Inflection Biosciences, Ltd), against CLL cells. In the current study we investigated the effects of combining IBL-202 with Venetoclax on primary CLL cells and both wild-type and TP53 deficient OSU-CLL cells.MethodsPrimary CLL cells were co-cultured with CD40L-expressing fibroblasts. We established a TP53 knock-out OSU-CLL cell line (OSU-TP53ko) using the CRISPr-Cas9 system. Cell viability was assessed using the mitochondrial dye DilC1(5), propidium iodide and flow cytometry. Synergy between IBL-202 and Venetoclax was evaluated by determining combination indices (CI) using the Compusyn software. The effects of the drugs on cell cycle and proliferation of the OSU-CLL cell lines were assessed using propidium iodide or carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. The effects of the drugs on the migratory capacity of CLL cells were assessed by determining changes in CXCR4 expression and CLL-cell migration along an SDF-1a gradient. The mechanisms of action of the drugs were investigated by immunoblotting.ResultsIBL-202 and Venetoclax were highly synergistic against primary CLL cells co-cultured with CD40L-fibroblasts, with a CI of 0.4 at a fractional effect of 0.9 (Figure A and B). Synergy between the drugs was consistent with a significant (P < 0.05) reduction in the IC50 for both drugs.Synergy was also observed against wild-type (WT) and TP53ko OSU-CLL cells, with CI values of < 0.5 at fractional effects of 0.5 and 0.9. Synergy was consistent with significantly greater cytotoxic effects of the drugs in combination (Figure C. WT : P = 0.002 and TP53ko : P = 0.002). IBL-202 and Venetoclax in combination induced cell cycle arrest and slowed the proliferation of both cell lines.Immunoblotting of primary CLL cells showed IBL-202, alone and in combination with Venetoclax, inhibited AKT phosphorylation and reduced the expression of Mcl-1 and Bcl-xL.A greater than additive effect of IBL-202 and Venetoclax was observed on the migratory capacity of CLL cells, reducing the number of cells migrating towards SDF1-a. The effects of the drugs on cell migration were consistent with reduced expression of CXCR4.ConclusionsThe synergy we observed between IBL-202 and venetoclax against primary CLL cells cultured under conditions that mimic the tumor microenvironment suggests this drug combination may be effective against CLL cells within the lymph nodes and bone marrow. Furthermore, the efficacy of the combination against the TP53ko OSU-CLL cell line suggests the combination may be a highly effective treatment strategy for poor risk CLL disease.Figure A and B - Synergy of IBL-202 and Venetoclax against primary CLL cells.Figure C - Cytotoxic effects of IBL-202 in combination with Venetoclax against OSU and OSU-TP53ko CLL cells [Display omitted] DisclosuresO'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees.