CLL Proliferation Research Articles

T‐helper cell regulation of CD45 phosphatase activity by galectin‐1 and CD43 governs chronic lymphocytic leukaemia proliferation

SummaryChronic lymphocytic leukaemia (CLL) is characterised by malignant mature‐like B cells. Supportive to CLL cell survival is chronic B‐cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to T‐helper (Th) cells in a CD40L‐dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non‐malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient‐derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation. Concomitantly increased was the surface expression of Galectin‐1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin‐1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin‐1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin‐1 and CD43‐mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL.

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Ibrutinib and Obinutuzumab in CLL: MRD Responses Sustained for Several Years with Deepest MRD Depletion in Patients with >1 Year Prior Ibrutinib Exposure

Ibrutinib and Obinutuzumab in CLL: MRD Responses Sustained for Several Years with Deepest MRD Depletion in Patients with >1 Year Prior Ibrutinib Exposure

The association of germline variants with chronic lymphocytic leukemia outcome suggests the implication of novel genes and pathways in clinical evolution

BackgroundChronic Lymphocytic Leukemia (CLL) is the most frequent lymphoproliferative disorder in western countries and is characterized by a remarkable clinical heterogeneity. During the last decade, multiple genomic studies have identified a myriad of somatic events driving CLL proliferation and aggressivity. Nevertheless, and despite the mounting evidence of inherited risk for CLL development, the existence of germline variants associated with clinical outcomes has not been addressed in depth.MethodsExome sequencing data from control leukocytes of CLL patients involved in the International Cancer Genome Consortium (ICGC) was used for genotyping. Cox regression was used to detect variants associated with clinical outcomes. Gene and pathways level associations were also calculated.ResultsSingle nucleotide polymorphisms in PPP4R2 and MAP3K4 were associated with earlier treatment need. A gene-level analysis evidenced a significant association of RIPK3 with both treatment need and survival. Furthermore, germline variability in pathways such as apoptosis, cell-cycle, pentose phosphate, GNα13 and Nitric oxide was associated with overall survival.ConclusionOur results support the existence of inherited conditionants of CLL evolution and points towards genes and pathways that may results useful as biomarkers of disease outcome. More research is needed to validate these findings.

Microenvironment-induced CD44v6 promotes early disease progression in chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) outgrowth depends on signals from the microenvironment. We have previously found that in vitro reconstitution of this microenvironment induces specific variant isoforms of the adhesion molecule CD44, which confer human CLL with high affinity to hyaluronan (HA). Here, we determined the in vivo contribution of standard CD44 and its variants to leukemic B-cell homing and proliferation in Tcl1 transgenic mice with a B-cell–specific CD44 deficiency. In these mice, leukemia onset was delayed and leukemic infiltration of spleen, liver, and lungs, but not of bone marrow, was decreased. Competitive transplantation revealed that CLL homing to spleen and bone marrow required functional CD44. Notably, enrichment of CD44v6 variants particularly in spleen enhanced CLL engraftment and proliferation, along with increased HA binding. We recapitulated CD44v6 induction in the human disease and revealed the involvement of MAPK and NF-κB signaling upon CD40 ligand and B-cell receptor stimulation by in vitro inhibition experiments and chromatin immunoprecipitation assays. The investigation of downstream signaling after CD44v6-HA engagement uncovered the activation of extracellular signal-regulated kinase and p65. Consequently, anti-CD44v6 treatment reduced leukemic cell proliferation in vitro in human and mouse, confirming the general nature of the findings. In summary, we propose a CD44-NF-κB-CD44v6 circuit in CLL, allowing tumor cells to gain HA binding capacity and supporting their proliferation.

Targeting BCR-Independent Proliferation of CLL Cells

The clinical success of PI3K and Btk inhibitors that block upstream BCR signals, has been primarily attributed to inhibition of adhesion and migration of CLL cells. However, the effects of these inhibitors on the proliferative capacity of CLL cells is largely unknown. Interestingly, in vitro BCR triggering does not induce proliferation of CLL cells. Instead, we have found that activated T cell-derived signals, specifically CD40L and IL-21, initiate CLL proliferation in vitro and can be traced in CLL lymph node samples from patients [Pascutti et al., Blood 2013]. Targeting the underlying signaling pathways may be clinically relevant, and therefore we aimed to characterize the molecular mechanisms underlying antigen-independent proliferation.In the present study we applied CD40L/IL-21 stimulation to induce proliferation, and tested various specific kinase inhibitors for blocking potential. Surprisingly, the Btk inhibitor ibrutinib significantly suppressed CD40L/IL-21-induced proliferation (Figure 1). Similarly, inhibition of PI3Kδ with idelalisib dampened proliferation. While neither ibrutinib nor idelalisib affected CD40L-induced NF-kB or IL-21 induced STAT3 phosphorylation, both compounds efficiently blocked downstream ERK1/2 phosphorylation. Impact of idelalisib and ibrutinib was non-synergistic, suggesting that PI3K and Btk are acting in concert. In contrast with the inhibitory effects of the two compounds above, no effect of CD40L/IL-21 induced proliferation was observed following inhibition of SYK activity with R406 (tamatinib). This strongly suggests a role for PI3K and Btk signaling independent of BCR-signaling.We demonstrate that antigen-independent proliferation completely depends on downstream CDK4-activity, which can be blocked by the CDK4-inhibitor PD0332991.IL-21 signaling is absolutely required for antigen-independent proliferation as treatment with the pan-JAK inhibitor INCB abrogates proliferation. Moreover, immunohistochemical staining of lymph nodes from CLL patients indicated the presence of phospho-STAT3 in CLL cells at lymph node sites, suggesting a contribution of JAK/STAT signaling to in vivo proliferation.In conclusion, our data strongly suggest that non-BCR signals contribute to proliferation of CLL cells. Since the most recent clinical data point to development of resistance against ibrutinib, targeting non-BCR pathways may offer additional venues for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Serum chemokines and cytokines in CLL patients treated with duvelisib, a PI3K-δ,γ inhibitor.

7072 Background: PI3K-δ and PI3K-γ play complementary roles in malignant B-cells and the tumor microenvironment (TME). Inhibition of PI3K-δ blocks cytokine-mediated CLL proliferation, while inhibition of PI3K-γ blocks M2 macrophage polarization and T-cell migration in vitro. Duvelisib, an oral PI3K-δ,γ inhibitor, has shown clinical activity in a phase 1 study in patients (pts) with advanced hematologic malignancies (Study IPI-145-02), including pts with relapsed/refractory (R/R) CLL (O’Brien, ASH 2014). Methods: Serum from 52 pts with R/R CLL and 30 healthy subjects (HS) was analyzed for 72 analytes (cytokines, chemokines, and matrix metalloproteinases) using Luminex xMAP technology at baseline, Cycle 1 Day 8 (C1D8), C2D1, and C3D1. Median change from baseline was analyzed for statistical significance using a paired t-test with Bonferroni correction for multiple hypotheses; threshold for reduction was ≤ 50% of baseline and for increase was ≥ 150% of baseline. Comparison between CLL and HS utilized a 2-sample t-test with Bonferroni correction. Results: Following treatment with duvelisib, the median serum levels of 12 analytes decreased to ≤ 50% of baseline (p < 0.0002) by C1D8. These included CCL1, CCL3, CCL4, CCL17, CCL22, CXCL10, CXCL13, IL-6, IL-10, IL-12p40, MMP-9 & TNFα. All of these were significantly elevated (p < 0.0042) at baseline in CLL pts compared to HS and reduced towards normal range following duvelisib treatment. In addition, MMP1 was the only analyte for which median serum levels increased above the threshold of ≥ 150% of baseline (p < 0.0002). Together, these 13 analytes were further explored for potential associations with clinical efficacy. TNFα was significantly elevated (p < 0.0013) at C2D1 & C3D1 in pts who did not exhibit a nodal response. Conclusions: Most of the analytes reduced following duvelisib treatment are known to be involved in the communication between CLL cells and the TME. Furthermore, one of these analytes (TNFα) exhibited elevated levels at C2D1 and C3D1 in pts who did not achieve a nodal response. Together, these data indicate that modulation of the TME via PI3K-δ,γ inhibition may be an important mechanism of action in supporting clinical activity of duvelisib in patients with CLL. Clinical trial information: NCT01476657.

Combined Inhibition of Phosphatidylinositol 3-Kinase (PI3K) Isoform α and δ By the Pan-Class I PI3K Inhibitor SAR245409 (XL765) in Primary Chronic Lymphocytic Leukemia Cells Blocks Survival, Adhesion and Proliferation

CLL cells are highly dependent on B- cell receptor (BCR) signaling and on stimuli from the microenvironment for survival and proliferation. New drugs targeting PI3K downstream of BCR signaling have emerged as promising treatment options for patients with CLL. Among four PI3K catalytic subunits, the PI3Kd isoform is crucial for downstream BCR signaling, but the relative importance of the PI3Kα isoform in CLL is less clear. Impressive clinical activity of idelalisib in CLL and indolent NHL patients was recently reported. Idelalisib, a PI3Kd specific inhibitor, inhibits chemotaxis and adhesion of leukemia cells, resulting in rapid lymphocytosis followed by a decrease in lymphadenopathy. However, idelalisib has no direct impact on leukemic cell survival [1], raising the potential risk of residual clones responsible for the development of resistance. In this study, we evaluated the impact of a pan-class I PI3K inhibitor (SAR245409/XL765), a PI3Kα-specific inhibitor (BYL719) and a PI3Kd specific inhibitor (idelalisib) on PI3K/mTOR signaling, apoptosis, cell adhesion, CD40-induced survival and proliferation in primary patient derived leukemic cells.Phosphorylation of the downstream effector of mTOR, S6RP, was completely blocked by SAR245409 but not by BYL719 or idelalisib. SAR245409 induced apoptosis in unstimulated CLL cells (IC50= 0.86µM) in contrast to BYL719 or idelalisib (IC50 >10µM), demonstrating that targeting multiple PI3K isoforms is required to completely block the PI3K/Akt/mTOR pathway (table 1). Importantly, SAR245409 also induced apoptosis in p53 or ATM dysfunctional CLL samples. SAR245409, as well as idelalisib, and in contrast to BYL719 completely inhibited BCR-mediated adhesion to fibronectin [2]. Similarly, SAR245409 inhibited CD40L-mediated survival [3], and induced upregulation of the pro-apoptotic protein BIM. All 3 PI3K inhibitors inhibited CD40 ligation + IL-21-mediated CLL proliferation [4].This study revealed that the pan-class I PI3K inhibitor SAR245409 is more cytotoxic to primary CLL cells than PI3Kα or PI3Kd specific inhibitors. Furthermore, combined inhibition of PI3Kα and d can block signaling pathways that are critical for CLL survival, adhesion and proliferation in the LN microenvironment (see table 1). This work provides a rationale for the evaluation of SAR245409 in CLL patients either as monotherapy or in combination therapies.[1] Hoellenriegel et al. The phospoinositide 3′-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood 2011;(118):3603-3612[2] de Rooij et al. The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 2012;(119):2590-2594.[3] Smit et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;(109):1660-1668.[4] Pascutti et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019.Table 1The effect of the PI3Kd inhibitor idelalisib, PI3Kα inhibitor BYL719 or pan PI3K inhibitor SAR245409 on CLL cells in functional assaysPI3Kd inhibitorPI3Kα inhibitorpan PI3K inhibitorCytotoxicity (IC50)1>10µM>10µM0.86µMInhibition of adhesion248%**21%43%**ActivationInhibition of CD40L-induced survival314%0%54%*Inhibition of CD40L+IL21 induced proliferation447%*35%*51%*1 CLL cells were incubated with 0.001-10 μM idelalisib (n=18), BYL719 (n=6) or SAR245409 (n=28) for 48 hours. Viability was assessed by DiOC6/PI staining.2 CLLcells pretreated with 1 µM idelalisib, BYL719, or SAR245409 were stimulated with α-IgM and allowed to adhere to fibronectin-coated surfaces (n=5). 3 CLL cells were cultured on fibroblast expressing CD40L in the absence or presence of 1 µM of idelalisib, BYL719, or SAR245409 for 3 days. Apoptosis was assessed by DiOC6/PI staining (n=8).4 CFSE labelledCLL cells were cultured on fibroblast expressing CD40L with IL-21 and co-treated with 1 µM idelalisib, BYL719, or SAR245409. After 4 days, CFSE was measured by FACS (n=11)2-4 The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01 DisclosuresEgile:Sanofi: Employment. Kersten:Sanofi: Research Funding. Kater:Sanofi: Research Funding.

Microrna Mir-21 Is Upregulated after Different Microenvironmental Stimuli and Controls Proliferation, Chemotaxis and Chemoresistance in Chronic Lymphocytic Leukemia

Aberrant expression of diverse microRNAs (miRNAs) has been related to pathogenesis and clinical outcome in patients with CLL. miR-21 is overexpressed in a wide variety of neoplasms where it participates in oncogenic events such as proliferation, resistance to treatment and metastasis. In CLL, miR-21 expression has been associated to refractoriness to fludarabine and to shorter overall survival. In addition, high expression of ZAP-70 protein in CLL is related to shorter overall survival and higher probability of progression. Several biological mechanisms have been described explaining the adverse prognosis associated with high ZAP-70 expression. In this sense, ZAP-70 protein increases the capability of CLL cells to respond to different survival and migration signals provided by the microenvironment. In a previous work, we found that transfected B-cell lines with a ZAP-70 expressing vector (Calpe at al, Blood 2011) had an increased expression of several molecules, including miR-21. Against this background, we studied the relationship between ZAP-70 protein and miR-21. For this, we firstly analyzed the correlation of ZAP-70 with miR-21 in primary CLL cells from 32 patients. In this series we observed that miR-21 expression analyzed by QRT-PCR was significantly higher in patients with high expression of ZAP-70 compared to patients with low expression of ZAP-70 (mean miR-21 in high ZAP-70 (N=17) = 5.781 ± 1.517; mean miR-21 in low ZAP-70 (N=15) = 0.6783 ± 0.2730; p=0.0082). In order to further analyze the molecular mechanisms regulating the induction of miR-21 and the potential role of ZAP-70 protein in this process, we co-cultured primary CLL cells (N=16) in conditions mimicking the microenvironment in the proliferation centers (bone marrow stromal cells with concomitant stimulation of CD40 and TLR9). In these conditions, besides ZAP-70 activation, we observed a 3.6 ± 0.78 mean fold induction in miR-21 expression after 48 hours compared to cells in suspension. Interestingly, this increase was only significant in patients with high expression of ZAP-70 (N=8; p= 0.0379). To define the role of ZAP-70 in miR-21 regulation, we stably transfected Ramos B-cells with ZAP-70 protein and found that both MAPK and STAT3 participate in the induction of miR-21 expression after ZAP-70 activation upon BCR crosslinking. Moreover, using this system we observed downregulation of the tumor suppressors PTEN, PDCD4 and PIAS3, all of which have been found to be targeted by miR-21 in malignant cells. Next, we aimed to study the functional role of miR-21 in primary CLL cells co-cultured in conditions mimicking the microenvironment from the proliferation centers. For this, we analyzed survival, proliferation and response to fludarabine after inhibition of miR-21 expression by transfecting primary CLL cells with antisense miR-21 inhibitor. The co-culture of primary CLL cells significantly increased their survival after 48 hours regardless the inhibition of miR-21. However, the induction of proliferation (measured as percentage of Ki-67 positive cells) was significantly inhibited by the suppression of miR-21 (N=13; p=0.022). Next, the analysis of a small pilot cohort showed a consistent but not yet significant overcoming of the chemoresistance induced by the co-culture after the inhibition of miR-21 Interestingly, the sensitivity to fludarabine of primary CLL cells cultured in suspension was also increased by the inhibition of miR-21. Finally, we examined the ability of primary CLL cells to migrate towards CXCL12 and observed that inhibition of miR-21 resulted in a 2.62-fold reduction of the migration index (ratio of cells migrating towards culture media to cells migrating towards media with 200ng/mL CXCL12). In conclusion, we have showed the correlation and participation of ZAP-70 protein in the regulation of miR-21. We have also observed that miR-21 contributes to CLL proliferation, resistance to fludarabine, and chemotaxis towards CXCL12. Although further experiments are warranted in order to fully elucidate the regulation and functional role of miR-21 in CLL, these results help to enlighten the biology behind the adverse clinical outcome of patients with CLL and high expression of ZAP-70, and could potentially be exploited for the development of new treatments in a near future. DisclosuresBosch:Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Abstract 5182: Identifying differential gene expression and splicing events in chronic lymphocytic leukemia patients through whole transcriptome profiling

Abstract Chronic lymphocytic leukemia (CLL) is a cancer in which B-lymphocytes proliferate in an unchecked manner and escape normal apoptotic death. It is one of the most common leukemias in the Western world, and currently, there is no cure for CLL due to the heterogeneous ways in which it can proliferate. Due to the dynamics of how CLL can develop, it is very critical to investigate the oncogenic mechanisms that allow particular clinical CLL subtypes to expand, as well as discovering potential underlying mechanisms that are common among all CLL patients. With this comprehensive goal in mind, we investigated the gene expression landscape in CLL by performing whole transcriptome analysis via RNA sequencing in 47 CLL patients and 5 normal CD19+ B-cell donors to determine differential expression and isoform patterns in CLL. Between CLL and normal CD19+ B-cells, there were 725 differentially expressed genes (FDR p&amp;lt;.01), with 217 of these genes being upregulated in CLL. Several of these upregulated genes include CTLA4, CD276, CLNK, and PDCD1, all of which are involved in T-cell interactions. We also discovered significant upregulation in the kinase suppressor of Ras 2 (KSR2) gene, an event previously not reported in CLL. We also see clear clinical separation of IGHV unmutated vs. IGHV mutated CLL patients based solely on the expression signature created by hierarchical clustering. Examples of prognostic genes that correspond to IGHV status include previously identified genes LPL, LDOC1, PTCH1, and CRY1, as well as potentially novel prognostic expression markers SPG20, ARSD, KLK2, and MTSS1. We also utilized the SpliceSeq algorithm software and investigated the differential isoform patterns that take place between CLL patients and normal B-cell donors. Based on the results, we discovered 300 alternative promoter, 135 alternative terminator, 287 exon skipping, and 30 premature stop events that differ between CLL and normal B-cells. From these results, we validated two isoform switching events that take place in the splicing machinery genes SF3B1 and SNRNP70. For SF3B1, a short transcript devoid of the critical protein domains is overexpressed in CLL compared to the normal B-cell counterpart. For SNRNP70, a particular transcript transcribed by an alternative promoter is overexpressed in CLL patients compared to normal CD19+ B-cells. This overexpressed SNRNP70 isoform does not contain the RNA recognition motif crucial for 5′ RNA binding and splicing initiation, thus theoretically rendering its canonical action nonfunctional. Overall, transcriptome profiling of CLL patients using RNA sequencing allowed for discovery of differential expression changes previously not reported in CLL, as well as discovering novel splicing events that provide insight into potentially new oncogenic mechanisms in CLL proliferation. Citation Format: Austin Y. Shull, Junfeng Luo, Jeong-Hyeon Choi, Lirong Pei, Farrukh T. Awan, Eun-Joon Lee, Jimei Liu, Phillip J. Buckhaults, Xiao-Jie Yan, Nicholas Chiorazzi, Huidong Shi. Identifying differential gene expression and splicing events in chronic lymphocytic leukemia patients through whole transcriptome profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2014-5182

Targeted Treatments: Pre-Clinical Evaluation Of Efficacy By Tracking Clonal Diversity In CLL Xenograft Models

Progressive CLL with DNA damage response defects caused by ATM or TP53 inactivation is still a therapeutic problem. There is currently a growing appreciation of the subclonal complexity in this type of leukaemia, mostly supported by whole genome analyses of CLL samples such as SNP arrays and next generation sequencing, both highlighting the role of subclonal variations in progression and therapeutic response. Although revealing, these analyses do not inform about the clonal make-up of individual proliferating cells and such information might be necessary to determine effectiveness of novel targeted treatments.In order to determine and follow the clonal evolutionary millieu in individual CLL cases we developed multi-colour fluorescence in situ hybridization (MCF) to analyse CLL samples at the single cell level. We screened 134 samples for the presence of 11q and 17p deletions of which 25 were identified with one of these cytogenetic defects and these were screened alongside 28 samples with normal 17p and 11q loci for 13q and 6q deletion as well as trisomy 12. We found that all but four of the samples with 11q or 17p deletions had at least one other genomic abnormality, whilst only two of the 28 samples with normal 17p and 11q loci harboured two genomic abnormalities. We subsequently performed MCF on the 27 samples with multiple genetic abnormalities and generated evolutionary trees for each of the samples. Two types of clonal evolution were identified: linear and branched, with the latter being the more common. We were able to analyse eleven of these samples post-treatment and found that whilst the clonality of some samples was largely unaffected by treatment, others showed treatment-induced differences in the subclonal make-up. Furthermore, some samples also exhibited signs of evolution with the generation of novel subclones upon treatment.Pre-clinical testing of novel therapeutic agents in xenograft models requires that the subclonal architecture of engrafted samples is representative of the donor sample. To this end, modifications were made to two primary CLL xenograft models. Firstly, samples from three different CLLs with a complex karyotype and multiple subclones were engrafted with autologous ex-vivo stimulated T-cells into NOG mice. All CLL subclones displayed a capacity to engraft and proliferate in this xenograft model. Furthermore, the MCF protocol was implemented to assess subclonality in vivo. Mice were randomised to Rituximab or control saline treatment three times over a period of five days. Analysis of engrafted cells in the spleen a week later displayed a good response to Rituximab with a significantly lower number of hCD45+ CD19+ CD5+cells. However, upon isolation of CLL cells by FACS and assessment of clonal architecture using MCF it was shown that Rituximab treatment affected all subclones with a differential subclonal response. This resulted in a greater proportion of the CLL subclones with the greatest genetic complexity harbouring both 11q and 6q deletions still remaining.Finally, to recapitulate patient response to therapy, T-cell depleted, pre-treatment PBMC from a patient poorly responsive to bendamustine + Rituximab were engrafted in humanised mice. As in the patient, bi-weekly therapy for 3 weeks resulted in a limited, poor response to therapy with a corresponding small reduction of CFSE-labelled CLL PBMC and hCD45+ CD19+ cells.We conclude that the majority of CLLs display a branched pattern of evolution and that the subclone dynamic is an important determinant of CLL proliferation and response to treatment. We suggest that novel targeted therapies should be tested in the context of their ability to eradicate CLL subclones with the highest proliferative capacity, as these subclones are most likely to evolve. The development of an MCF protocol, in combination with the xenograft model, provides a powerful tool to help predict overall and subclonal responses to therapy in the patient. Disclosures:No relevant conflicts of interest to declare.

Evaluation Of CLL Adhesion Molecule Expression As a Diagnostic Tool For Disease Progression

IntroductionChronic lymphocytic leukemia (CLL) is a chronic B cell lymphoproliferative disorder characterized by small mature neoplastic lymphocytes, which primarily involve blood, bone marrow, and/or lymph nodes. Complex cellular and molecular interactions between CLL cells and the bone marrow or lymph node have been shown to affect CLL proliferation, survival and confer drug resistance that may be responsible for residual disease after conventional therapy. The standard methods used to assess CLL patient survival and treatment requirements involve the Rai and Binet staging systems. Pathologic markers such as CD38 expression by flow cytometric analysis, immunoglobulin heavy chain variable region (IgVH) mutation status, and conventional cytogenetics also demonstrate loose association with overall patient survival. However, these systems cannot identify stable or progressive forms of the disease in individual patients, especially in the early stages of CLL. ObjectiveThe objective of this study is to evaluate the expression profile of specific adhesion molecules that may regulate critical interactions between CLL cells and the bone marrow as potential dynamic biomarkers that correlate with CLL disease progression and response to chemotherapy. We evaluate the co-surface-expression of the molecular scaffold protein, CD82, with its interacting integrin partner, alpha 4 on CLL cells. We correlate the expression of these adhesion molecules with the patient's clinical stage, lymphocyte doubling time (LDT), IgVH mutation status, conventional cytogenetics and the expression level of CD38 antigen on CLL cells. MethodsA single-institution study prospectively enrolled CLL patients diagnosed and followed up at a university-based cancer center. Peripheral blood samples were obtained from consented patients at the time of their follow up appointments. Peripheral blood mononuclear cells were isolated from the whole blood using a ficoll density gradient and CLL cells were isolated by magnetic bead negative selection. Using flow cytometry, CLL cells were analyzed for alpha 4 and CD82 surface expression and the median fluorescence intensity values were correlated with the patient's clinical staging parameters (Ann Arbor system, Rai stage classification) and LDT. A non-parametric measure (Spearman's rho) was used to assess the correlation between variables. The correlation analyses were stratified by indolent cases versus aggressive disease cases and, also by the IgVH mutation status and the presence of CD38 overexpression and cytogenetics at the time of diagnosis. An unpaired t-test was used to compare differences in the CD82 and alpha 4 expression among groups. ResultsAt the time of abstract submission, a total of 18 patients were enrolled in the pilot study. Nine patients had indolent disease and had not received treatment for CLL, whereas nine patients had received treatment for CLL at the time of enrollment. CD82 and alpha 4 expression data were not obtained for three patients on active treatment due to severe lymphopenia. CD82 and alpha 4 data were obtained for all of the indolent cases. IgVH mutation status and CD38 expression data at diagnosis were evaluated when available. A statistically significant correlation (P< 0.05) was observed for the co-expression of CD82 and alpha 4 in all of the cases. When analyzed by subgroups, the co-expression of CD82 and alpha 4 remained statistically significant in the indolent disease group and there was a similar trend in the aggressive disease group that did not reach statistical significance. In the aggressive disease group, CD82 expression negatively correlated (P<0.05) with lower absolute lymphocyte counts and a lower platelet counts at the time of enrollment in the study. In addition, we observed a trend of negative correlation between the CD82 expression and LDT in the indolent disease group. For those patients with CD38 overexpression a trend to a higher expression of CD82 and alpha 4 was found. ConclusionIn this initial data set, we observed trends of correlation of CD82 and alpha 4 integrin expression with platelet count, absolute lymphocyte count, LDT and CD38 overexpression. In future work, we will continue to recruit more patients to enhance the statistic power of the study. Furthermore, we will evaluate the expression level changes of CD82 and alpha 4 during treatment and their role in response to chemotherapy and CLL metabolic activity. Disclosures:No relevant conflicts of interest to declare.

Proliferating CLL Cells Express Abundant But Transcriptionally Compromised TP53 Protein

Current models of CLL pathogenesis invoke specialized microenvironments within the lymph nodes and bone marrow that harbor proliferating cells. Such proliferating CLL cells are more resistant to current immuno-chemotherapeutic regimens than cells in the peripheral blood and are thought to be the cause of resistant disease. Various models have been used to recapitulate these CLL proliferation centers in vitro, including stimulating cells with CD154 and IL4. We studied 40 patients (6 with p53 mutations/deletions) using this system and observed that >50% of CLL cells undergo proliferation after 72 hours of stimulation, as assessed by Ki67 staining. Unexpectedly, under these conditions we also observed a 30-40 fold induction of TP53 protein in all cases of CLL analyzed, irrespective of TP53 mutational status. Nearly all cells showed increased TP53 protein expression (Figure 1), suggesting that such high-level TP53 protein expression did not hinder cell proliferation. Given that induction of wild-type TP53 protein usually induces cell cycle arrest if not apoptosis, we examined for transcriptional up-regulation of TP53 target genes using a combination of qRT-PCR, RNA arrays and RNA-Seq approaches. 4 out of 12 cases showed induction of some TP53 target genes, but overall there was no consistent pattern of transcriptional up-regulation of target genes, suggesting that the induced TP53 is transcriptionally compromised in CLL cells following CD154/IL4 stimulation. In contrast, DNA damage induced by doxorubicin in CD154/IL4 stimulated cells induced wild type TP53 protein to even higher levels, resulting in TP53 target gene up-regulation and apoptosis, as expected. CD154/IL4 stimulation also induced a 10-fold elevation in ROS levels in all cases. This resulted in significant oxidative DNA damage, as measured by a modified comet assay, which could explain the induction of TP53 in proliferating cells. qRT-PCR and RNA-Seq experiments failed to show a significant increase in TP53 mRNA levels, indicating that elevation of TP53 protein levels was occurring post-transcriptionally. Increased phosphorylation of TP53 at S15 was seen in all cases, which may account for the observed increased protein stability through dissociation from MDM2. All TP53 mRNA isoforms expressed retained transcriptional activation and DNA binding domains. In view of these results, we propose a model whereby oxidative stress induced by proliferation in CLL triggers TP53 protein expression. TP53 becomes phosphorylated but, for reasons that remain unclear, is unable to transactivate its target genes normally and induce cell-cycle arrest. Apoptosis could be suppressed by high-level expression of anti-apoptotic BCL2 proteins. However, TP53 remains able to trigger a full apoptotic response after further DNA damage and a higher threshold of protein levels is reached. Reactivation of the full transcriptional activities of wild-type TP53 in proliferating CLL cells may provide a new therapeutic approach. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Stromal Tumor Microenvironment in CLL: Regulation of Leukemic Progression

AbstractAbstract 1781Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patients’ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

BTK Inhibition Targets in Vivo CLL Proliferation Through Its Effects On B-Cell Receptor Signaling Activity.

AbstractAbstract 2903 Purpose:Bruton tyrosine kinase (BTK) is a component of the B-cell receptor signaling pathway. Ibrutinib (previously known as PCI-32765), a first in class, covalent BTK inhibitor, has demonstrated significant clinical activity against CLL in early clinical trials. Understanding the molecular mechanisms of action would shed light on CLL pathophysiology and provide additional opportunities for the development of new therapies. Experimental Design:The anti-tumor activity of ibrutinib in CLL has been investigated previously using either an ex vivo approach or a mouse model (Herman et.al, Blood. 2011;117:6287–96 and Ponader et.al, Blood. 2012;119:1182–9). In this study, we have chosen, instead, a patient-oriented in vivo approach by using samples from an ongoing phase 1b trial of ibrutinib (NCT01105247). We prospectively collected serial samples from CLL patients (n=14) before and at several time points after the initiation of therapy and analyzed them for cellular and molecular signaling events. Results:We demonstrated that levels of the phosphorylated BTK protein (p-BTK) in CLL cells from treatment-naïve patients were significantly higher than in normal B cells, explaining why CLL cells are more susceptible to BCR inhibition than normal B cells. Response assessments, performed at the end of cycle 2 (∼Day 56), demonstrated nodal responses in all patients by CT scan. Ex vivo apoptosis did occur but required high concentrations of ibrutinib (>500 nM). In addition, in vivo apoptosis was rarely observed in serial peripheral blood samples collected from treated patients. With these serial samples, we found that the population of Ki67+ cells were gradually decreased over a 28-day ibrutinib treatment course. Using a newly established co-culture system that induces CLL proliferation in vitro, the analysis of several parameters, including Ki-67 expression, cell growth and bromodeoxyuridine (BrdU) incorporation (shown in the figure), revealed that the proliferation of CLL cells was directly inhibited by ibrutinib (200 nM). Furthermore, activities of BTK and downstream signaling events, such as the phosphorylation of PLCg2, AKT and ERK, were all suppressed over time in ibrutinib-treated patients. Conclusions:With primarily an in vivo approach, we have demonstrated that the blockage of cell proliferation was a major effect of ibrutinib against leukemic CLL cells. Blocking cell proliferation via inhibition of BTK-mediated signaling concurs with clinical responses in ibrutinib-treated CLL patients. [Display omitted] Disclosures:Leonard:Pharmacyclics Inc.: Consultancy, Honoraria. Buggy:Pharmacyclics: Employment, Equity Ownership.

Primary CLL Xenograft: T-Cells Friend or Foe? the Role of T-Cells in the Primary CLL Xenograft Model.

AbstractAbstract 2888Chronic lymphocytic leukaemia (CLL) is a malignancy characterised by the gradual accumulation of mature B cells in peripheral lymphoid organs. However, limited access to this proliferating tumour population in CLL patients and difficulties in modelling this proliferation in vitro necessitates the establishment of human xenograft models that can recapitulate the human disease. Several CLL xenograft models have already been established, however due to their inability to fully recapitulate human tissue distribution and their restricted period of engraftment, these models are not suitable for testing novel chemotherapeutics.To address this, modifications were made to a recent xenograft model established by Bagnara et al, utilising immuno-compromised NOG mice on a human haematopoietic microenvironment provided by prior propagation of human CD34+ umbilical cord blood cells. This was reported to reliably replicate the human disease but was of limited duration purportedly due to T-cell outgrowth.Our aim therefore was to determine the role various T-cells subsets play in engraftment and its demise in order to enhance the duration of the CLL engraftment in this primary CLL xenograft model. This was accomplished, by a) sequential cull of animals engrafted by primary CLL cells to monitor kinetics of T-cell engraftment over time, b) by addressing clonality and the potential anti-CLL effect of expanded T-cells and c) by depletion of implicated autologous T-cell subsets prior to their co-administration into mice with CLL tumour cells.The engraftment kinetics of patient PBMC in murine spleen; encompassing both T-cells and tumour CLL; were tracked and we observed that the relative proportions of patient T-cell subsets altered within 8 weeks following injection. Initially, during the first 6 weeks, there was rapid expansion of helper (CD4+) and regulatory (CD4+/CD25+) T-cells. Subsequently, their proportion decreased as the cytotoxic (CD8+) T-cells expanded becoming the dominant T-cell subtype. Interestingly, by using PCR amplification of TCR rearrangements we noted that these T-cells were not clonally expanded and that their co-incubation with autologous CLL cells in vitro failed to exert a cytotoxic effect.We have previously demonstrated that CLL engraftment could occur even if patient T-cells were depleted to a minimal level of 0.6–5×104 cells or 2% of initial T-cell numbers. Here, we demonstrate that depletion of either helper (CD4+) or regulatory (CD25+) T-cells resulted in a reduced level of engraftment in murine peripheral blood and spleen, extended graft duration and prolonged survival of the animals. In contrast, depletion of either cytotoxic (CD8+) or natural killer (CD56+) T-cells had limited effect on both engraftment initiation and duration of the graft (none<CD8<CD56<CD4<CD25<CD3, 101 vs 96 vs 115 vs 127 vs 130 vs 151 days).Taken together our results indicate a crucial role for helper (CD4+) and regulatory (CD25+) T-cell subsets in both initiation and termination of CLL engraftment. The expansion of CD8+ T-cells at the end of engraftment may be co-incidental and not a reflection of an anti-tumour effect. Further studies are necessary to address the exact interaction between patient CLL cells and regulatory T-cells. Finally, manipulation of the regulatory T-cell subset in this xenograft model can provide robust CLL proliferation amenable for drug testing. Disclosures:No relevant conflicts of interest to declare.

The NSG – Human CLL Xenograft Model Recapitulates the Human Lymph Node Microenvironment in Regards to B-Cell Activation and Tumor Proliferation

Abstract 973Chronic lymphocytic leukemia (CLL) cells depend on signals from the tissue microenvironment for proliferation and survival. A comparative analysis of lymph node (LN) and peripheral blood (PB) derived CLL cells revealed dynamic B-cell receptor (BCR) and NF-kB activation in this microenvironment (Herishanu et al, Blood 2011). To dissect the pathogenic role of different signaling pathways in vivo, it is crucial to develop a model system that can reproduce the tumor biology encountered in the human LN. We hypothesized that secondary lymphoid compartments (such as the bone marrow (BM) or spleen) of mice may recapitulate the human LN microenvironment. In order to study the effect of the murine microenvironment on human CLL cells we chose to use the recently established NOD scid gamma null (NSG) - human CLL xenograft model (Bagnara et al., Blood 2011), with slight modifications: no adoptive transfer with normal human CD34 or CD14 positive cells was undertaken, and NSG mice were conditioned with 25 mg/kg busulfan instead of g-irradiation. 24 hours later, 1 × 108 CFSE labeled CLL PB mononuclear cells (PBMCs) were injected through the tail vein. Mice were bled weekly and sacrificed between two and six weeks post xenograft. The PB CLL cell count (evaluated as hCD45+, hCD19+, hCD5+ cells) one week after injection averaged 300 cells/μL but then rapidly declined and became virtually undetectable by week four. In contrast, CLL cells persisted in the spleen and BM. We next sought to determine if tumor proliferation was occurring in the xenograft model. We found that CLL proliferation (measured by the fraction of CFSE low cells) was barely apparent early (weeks 1–2) but increased significantly by weeks 3–4 (p=0.02). A similar trend was also observed for T-cells. There was no significant difference in the fraction of CFSE low cells among the three different compartments; however, the number of CLL cells in the spleen was significantly higher than in the PB or BM, suggesting increased homing to the spleen. We next assessed if the mouse spleen microenvironment would modulate the expression of CD38, CD69 and CD184 (CXCR4). Xenografted CLL cells extracted from murine spleens showed significantly increased expression of CD38 (p=0.01) and CD69 (p<0.001) and significantly decreased expression of CD184 (p<0.001) compared to PB cells, recapitulating findings previously reported in patients. We have previously shown that LN-resident CLL cells display gene expression signatures indicating BCR and NF-kB activation. We therefore sought to determine if the mouse spleen microenvironment would modulate gene expression in CLL cells. We compared patient derived PB-CLL cells to either matched patient derived LN-CLL cells or matched xenografted CLL cells isolated from the murine spleen and evaluated expression of BCR, NF-kB, and MYC target genes using quantitative RT-PCR (pre-designed Taqman Gene Expression assays). BCR (AICDA, EGR1, EGR3, GFI1, KLF10, OAS3) and NF-kB (CCL4, CCL3, RGS1, TNF, CCND2) target genes were upregulated in CLL cells from both the human LN and murine spleen. In addition, genes associated with proliferation (CDT1, PCNA, RRM2) were upregulated in CLL cells from both LN and murine spleen; consistent with the proliferation measurements using CFSE. However, MYC target genes (MYC, DUSP1 and HSPD1) were upregulated only in CLL cells from LN but not in the murine spleen. It has been hypothesized that blocking CXCR4 on tumor cells could inhibit their ability to home and interact with the tissue microenvironment. We therefore tested the effect of AMD3100 – a CXCR4 antagonist (5 mg/kg/day by continuous subcutaneous infusion) in this model. Unexpectedly, there was no effect on leukemic cell proliferation or on the CLL count in the PB of treated as compared to untreated mice. However, we found a dramatic increase in CXCR4 expression on CLL cells in the treated mice, indicating adequate drug delivery. Taken together, our results suggest that the murine spleen microenvironment can adequately recapitulate that of the human LN. These results provide further justification for the use of the NSG - human CLL xenograft model to study both the pathogenic mechanisms that contribute to disease progression in the tissue microenvironment and as a pre-clinical model for drug development and assessment.Supported by the Intramural Research Program of NHLBI Disclosures:No relevant conflicts of interest to declare.

Role of Microenvironment-Associated Chemokines and Cytokines for Binet Stage A CLL Patients Included in a Prospective Trial (CLL1 trial) of the German CLL Study Group (GCLLSG): sIl2Ralpha Is An Independent Predictor of Progression-Free Survival (PFS),

Abstract 3869 Background:In CLL, proliferation of the leukemic cell clone occurs in the bone marrow and lymphatic tissues rather than peripheral blood. In this microenvironment, CLL cells interact with accessory cells, such as T cells and CD68+ nurselike cells (NLCs) and display signs of B cell receptor (BCR) activation, suggesting that CLL proliferation is T cell- and BCR-driven. In addition, cytokines and chemokines secreted by leukemic cells, stromal cells and T cells are essential in forming the disease-specific microenvironment. However, the exact biological role of cytokines and chemokines needs to be defined, especially in the light of distinct prognostic and biological subgroups of patients (pts). Methods:In order to address this issue, we measured serum levels of chemokines and cytokines in a prospective cohort of 157 previously untreated Binet stage A pts., a small subgroup of the risk-stratified CLL1 trial of the GCLLSG. Median follow-up time of that subgroup was 50.3 months. Median time to progression was 61.3 months. Median time from diagnosis to study entry was 1 year. Serum samples had been centrally collected at study entry and stored at −80°C. Sera were analyzed on a luminex-based multiplex platform, allowing simultaneous screening of multiple serum parameters. In a pilot phase, sera of 21 pts were pre-analytically screened for a total of 62 different chemokines. Median serum levels of 27 different chemokines and cytokines were found to differ from healthy controls in a significant manner. Those 27 chemokines and cytokines were subsequently analyzed in 157 pts. For all parameters univariate and multivariate analyses were performed for progression-free survival. Results:Serum levels of CCL3 and CCL4, chemokines known to be secreted by CLL cells upon BCR engagement, were elevated compared to healthy controls. High CCL3 levels correlated strongly with high CCL4 levels (p<0.001) and tended to be associated with unmutated IgHV status (p=0.06), supporting the role of BCR triggering in biologically selected CLL pts. Concerning CCL3 and CCL4, no significant difference in PFS was detected. Serum levels of CCL2 and CCL17, both binding chemokine receptor CCR4, were concordantly elevated compared to healthy controls (p<0.001), suggesting a possible role of chemoattraction of CCR4+ T cells towards these chemokines in CLL. CXCL12, CCL21, and CXCL10, chemokines associated with chemotaxis of CLL cells, were concordantly elevated compared to healthy controls. Pts with serum levels of CCL21 beyond the median also had higher levels of CXCL10 (p<0.001) and CXCL12 (p=0.02). CLL pts have been found to have abnormal neovascularization in the bone marrow and lymph nodes. Elevated VEGF levels were strongly associated with elevated EGF level (p<0.001). High VEGF levels correlated with high white blood cell counts (p=0.008) and showed a trend towards association with del(11q) (p=0.07) and lymphadenopathy (p=0.165). Concerning VEGF, no significant difference in PFS was detected. In contrast, pts with high levels of sIl2R alpha showed a significant shorter PFS. When confirmed by conventional ELISA, median PFS in pts within the highest quartile of sIl2Ralpha levels were 22 months compared to 72 months in the lower three quartiles (p<0.001). When we analyzed sIl2R alpha together with genetic abnormalities like del(11q), trisomy 12, del(13q), del(17p), the risk stratification model used in CLL1 (high risk versus low risk for disease progression), the hierarchical model as published by Döhner et al., and IgHV status in a multivariate Cox regression, we found that sIl2R alpha (OR 2.5, 95% CI 1.4–4.8, p=0.004) and IgHV mutational status (OR 4.0, 95% CI 2.2–7.3, p<0.001) were both independent prognostic variables. Conclusion:The assessment of sera in a small subgroup of the CLL1 study cohort of the GCLLSG revealed that the levels of several chemokines and cytokines were elevated when compared to healthy controls. Median serum levels of different chemokines correlated with distinct biological characteristics of CLL pts, like genetic abnormalities or clinical parameters. In addition, sIl2R alpha could be identified as an independent prognostic variable for PFS in early CLL. Disclosures:Eichhorst:Hoffmann La Roche: Honoraria, Research Funding, Travel Grants; Mundipharma: Research Funding, Travel Grants; Gilead: Consultancy. Stilgenbauer:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel Grants. Hallek:Hoffmann La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Bergmann:Celgene: Honoraria.

SYK Inhibition Targets CD38−Mediated Survival and Proliferation Signals in Chronic Lymphocytic Leukemia Cells

Abstract 800Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. The highly variable prognosis of the disease may be predicted using a number of biomarkers, including the expression level of CD38. Human CD38 is a surface glycoprotein with enzymatic activity and the ability to mediate cell-cell interactions by binding the non-substrate ligand CD31. CD31/CD38 interactions drive CLL proliferation and chemotaxis, and increase CXCL-12-mediated signals and homing of CLL cells towards lymphoid organs. We and others have previously identified SYK, a key component of the BCR signalling pathway, as a candidate for targeted therapy in CLL due to its enhanced expression and activity and the apoptotic effects of pharmacological SYK inhibition even in the context of the microenvironment. In this study, we demonstrate direct involvement of SYK in the CD38 signaling pathway and that SYK inhibition may prevent CD38−mediated CLL cell proliferation and migration.CD38 stimulation of primary CLL cells by its ligand CD31 resulted in SYK activation as indicated by phospho-protein analysis by flow cytometry (n=18, p=0.0002) and immunoblotting. Analysis of CD38 expression on CLL cells by flow cytometry with SYK inhibition using the pharmacological SYK inhibitor R406 (Sellek) revealed a significant decrease of CD38 by 31% (n=25, p<0.0001). Particularly, CLL cells with high CD38 expression showed a substantial downregulation following SYK inhibition. Since CD38 expression is associated with poor clinical outcome, the proliferative signals delivered by its ligation might be critical for this association. Therefore, we tested the influence of SYK inhibition on proliferative signals as assessed by the expression of the proliferation marker Ki-67. 12 hours of stimulation with CD31 resulted in up-regulation of Ki-67 in CLL cells, an effect completely abrogated by concomitant treatment with the SYK inhibitor R406 (n=8). Since the expression of CD38 is regulated by different pathways including CD40 ligation and SYK involvement has been implicated in CD40 signaling, we evaluated whether R406 is capable to inhibit CD38 upregulation induced by CD40L. Indeed, in vitro stimulation with 3 μg/ml recombinant CD40L for 30 hours resulted in a significant increase of CD38 expression (p=0.0059) and was reversed by concomitant SYK inhibition (p=0.0142, n=9). As positive control, we treated CLL cells with ATRA that upregulated CD38 in myeloid cells. ATRA is directly translocated to the nucleus without requirement of signal transduction. Hence, SYK inhibition did not influence ATRA-mediated CD38 upregulation. In order to functionally characterize the role of SYK in the CD38/CD31 interaction in CLL cells, we analyzed the expression of the anti-apoptotic molecule MCL-1 in the presence and absence of SYK inhibition with and without CD38 stimulation. CD31 ligation enhanced MCL-1 expression, an effect reversed when CLL cells were coincubated with R406 (n=8).Taken together, our data demonstrate direct involvement of SYK in the CD38 signaling pathway. Beyond the known effects of SYK inhibition on chemokine- and integrin mediated CLL-stroma interactions, these data establish interference with the CD31/CD38 axis as novel therapeutic mode of action of SYK inhibition in CLL. Since CD38 expression identifies CLL cells with high proliferative and migratory function and clinically associates with poor responsiveness to chemotherapy SYK inhibition may be a particularly promising therapeutic option in CLL patients expressing CD38. Disclosures:No relevant conflicts of interest to declare.

2.24 Sensitization of High-Risk Chronic Lymphocytic Leukemia Cells to Fludarabine Using Inhibition of DNA-Dependent Protein Kinase

to capture probe-bound Btk, followed by detection with an anti-Btk antibody. Results: AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 0.5 nM) and inhibits primary B-cell proliferation and activation (EC50 approximately 10 nM). AVL-292 inhibits NLC-derived survival of primary CLL cells and production of CCL3 and CCL4, chemokines that nucleate costimulatory immune cells to promote CLL proliferation and demonstrated biomarkers of BCR activation in CLL cells. In addition, AVL-292 blocks CLL cell migration towards CXCL12 and CXCL13, disrupts reorganization of the actin cytoskeleton in response to these chemokines, and probably further weakens the ability of these cells to remain anchored to the proliferative microenvironment necessary for growth and survival. An assay that enables direct measurement of Btk–AVL-292 interaction was used to correlate the degree of Btk covalent bonding with efficacy of AVL-292 in vitro and in vivo. Recent completion of an innovative human clinical trial with AVL-292 has demonstrated that a single dose of 1 to 2 mg/kg of AVL-292 is required to completely bond and inhibit the molecular target, Btk. Moreover, the rate of Btk protein resynthesis in these human subjects was determined to be approximately 50% resynthesis in 48 to 72 hours and supports a once-daily dosing schedule. Conclusions: Targeted covalent drug design has enabled the discovery and early clinical development of a potent and highly selective inhibitor of Btk, AVL-292, and supports Btk as an exciting new target for B cell cancers. These data demonstrate that specific targeting of Btk inhibits the survival of primary CLL cells and disrupts chemokine signaling pathways believed to be important in maintaining the CLL tumor microenvironment. The covalent mechanism of action of AVL-292 can deliver translational pharmacodynamics early in clinical drug development and has facilitated the rational selection of dose and dose schedule to maximize patient benefit in clinical trials studying B-cell cancers.