Potent Efficacy of Combined PI3K/mTOR and JAK or SRC/ABL Inhibition in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Background. Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with genomic alterations that activate JAK/STAT and PI3K/Akt/mTOR signal transduction and with poor clinical outcomes. Therapeutic disruption of PI3K pathway signaling in Ph-like ALL has been minimally investigated to date, however. We hypothesized that PI3K isoform-selective or dual PI3K pathway protein inhibition would robustly inhibit Ph-like ALL proliferation in vivoand abrogate aberrant signaling. Methods. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were engrafted with primary CRLF2/JAK-mutant or ABL/PDGFR-mutant Ph-like ALL specimens (Table 1) and treated with inhibitors of PI3K? (BYL719), PI3K? (idelalisib), PI3K/mTOR (gedatolisib), TORC1/TORC2 (AZD2014) or with vehicle. Treated patient-derived xenograft (PDX) models were assessed for pharmacodynamic inhibition of signal transduction phosphoproteins at 72 hours by phosphoflow cytometry and for residual ALL in murine spleens after 3-4 weeks of inhibitor or vehicle treatment by quantitative flow cytometry. Subsequent studies tested the efficacy of gedatolisib with the JAK1/2 inhibitor ruxolitinib (CRLF2/JAK-mutant models) or gedatolisib with the SRC/ABL inhibitor dasatinib (ABL/PDGFR-mutant models). Table 1. Genomic characteristics of Ph-like ALL specimens utilized for PDX studies. USI Disease status CRLF2/JAK alterations ABL/PDGFR alterations PALTWS D IGH@-CRLF2* PAMDKS D IGH@-CRLF2, JAK2R683G PAMDRM D IGH@-CRLF2,JAK2GPinsR683 ALL121 R IGH@-CRLF2, JAK2R683G ALL4364 R P2RY8-CRLF2, JAK2R683G PAKMVD D JAK1 S646F PAKYEP D BCR-JAK2 PAKKCA D EBF1-PDGFRB PAKVKK D NUP214-ABL1 PANSFD D ETV6-ABL1 USI = unique specimen identifier. D = de novo, R = relapse. * non-Ph-like by prediction analysis of microarrays. Results. All tested PDX models demonstrated inhibition of leukemia proliferation and abrogation of activated signaling with PI3K pathway inhibition. Gedatolisib treatment resulted in near-eradication of leukemia in CRLF2/JAK-mutant models (n=7) with mean 92.2% (range 86.0-99.4%) leukemia reduction vs vehicle treatment (p<0.0001), as well as prolonged animal survival (p<0.005). Gedatolisib also inhibited leukemia proliferation in ABL/PDGFR-mutant models (n=3) with mean 66.9 (range 42.0-87.6) leukemia reduction vs vehicle controls (p<0.0001). BYL719, idelalisib, and AZD2014 monotherapy decreased ALL burden in JAK-mutant models with mean 52.7% (range 27.5-72.9%), 41.6% (range 22.6-53.1%), and 56.3% (range 20.1-88.7%) reduction, respectively. These three inhibitors had variable potency in ABL/PDGFR-mutant models with 39.1% (range 11.4-71.2%) ALL reduction with BYL719, 0.4% (range -25.2-13.9%) reduction with idelalisib, and 14.5% (range -15.5-30.7%) reduction with AZD2014 treatment vs vehicle controls. Leukemias with greatest basal signaling activation measured by phosphoflow cytometry demonstrated greatest leukemia reduction with PI3K pathway inhibition in the treatment studies, suggesting that basal phosphoprotein levels may be predictive biomarkers of response. Given the recently reported efficacy of JAK inhibition in CRLF2/JAK-mutant and SRC/ABL inhibition in ABL/PDGFR-mutant Ph-like ALL PDX models, we hypothesized that dual pathway inhibition would be more effective than monotherapy. Indeed, simultaneous treatment with gedatolisib and ruxolitinib additively or synergistically reduced ALL burden in all tested CRLF2/JAK-mutant models vs gedatolisib or ruxolitinib monotherapy (p<0.001). Similarly, combined gedatolisib and dasatinib treatment induced markedly greater anti-leukemia efficacy in ABL/PDGFR-mutant models vs. either inhibitor alone (p<0.001). Conclusions. PI3K pathway inhibition is a biochemically relevant therapeutic approach for Ph-like ALL. Dual PI3K/mTOR inhibition with gedatolisib monotherapy potently inhibited leukemia proliferation and demonstrated additive or synergistic activity in combination with JAK or SRC/ABL inhibition in JAK-mutant or ABL/PDGFR-mutant Ph-like ALL, respectively. These data provide compelling rationale for testing combinations of signal transduction inhibitors without or with cytotoxic chemotherapy in children and adults with Ph-like ALL. Disclosures Off Label Use:preclinical testing of signal transaction inhibitors in Ph-like ALL models. Teachey:Novartis:Research Funding. Maude:Novartis:Consultancy, Research Funding. Perl:Actinium Pharmaceuticals:Consultancy; Asana Biosciences:Consultancy; Arog Pharmaceuticals:Consultancy; Ambit/Daichi Sankyo:Consultancy; Astellas US Pharma Inc.:Consultancy. Hunger:Sigma Tau:Consultancy; Jazz Pharmaceuticals:Consultancy; Spectrum Pharmaceuticals:Consultancy; Merck:Equity Ownership. Grupp:Novartis:Consultancy, Research Funding.

Elements of the B Cell Signalosome Are Differentially Affected by Mercury Intoxication

It has been suggested that environmental exposures to mercury contribute to autoimmune disease. Disruption of BCR signaling is associated with failure of central tolerance and autoimmunity, and we have previously shown that low levels of Hg2+interfere with BCR signaling. In this report we have employed multiparametric phosphoflow cytometry, as well as a novel generalization of the Overton algorithm from one- to two-dimensional unimodal distributions to simultaneously monitor the effect of low level Hg2+intoxication on activation of ERK and several upstream elements of the BCR signaling pathway in WEHI-231 B cells. We have found that, after exposure to low levels of Hg2+, only about a third of the cells are sensitive to the metal. For those cells which are sensitive, we confirm our earlier work that activation of ERK is attenuated but now report that Hg2+has little upstream effect on the Btk tyrosine kinase. On the other hand, we find that signaling upstream through the Syk tyrosine kinase is actually augmented, as is upstream activation of the B cell signalosome scaffolding protein BLNK.

Inhibition Of The Wnt Pathway Leads To Improved Chemosensitivity In Pediatric Acute Lymphoblastic Leukemia

Introduction While childhood acute lymphoblastic leukemia (ALL) is highly curable, up to 20% of children will relapse, with dismal prognosis, warranting the need for novel therapies. Previously, using an integrated genomic approach on matched diagnosis-relapse samples, we identified overactivation of the Wnt pathway as a mechanism of disease recurrence at relapse (Hogan et al, Blood 2011). Aberrant Wnt signaling has been linked to cancers of the liver, colon, breast, skin and more recently hematologic malignancies. To validate our findings and determine if Wnt inhibition could restore chemosensitivity in relapsed ALL, we sought to examine directly whether Wnt is activated at relapse in paired samples (examining expression of activated b-catenin and its downstream target Survivin (BIRC5) using multiparameter phosphoflow cytometry) and tested the efficacy of a recently developed small molecule Wnt inhibitor, iCRT14, that specifically interferes with the b-catenin-TCF interaction (Gonzalves et al, PNAS 2011), in ALL cell lines and patient samples. Methods B and T-ALL cell lines were treated with iCRT14 and the expression of target genes were determined by quantitative RT-PCR.10 paired diagnosis-relapse patient samples obtained from the Children’s Oncology Group were washed, fixed and stained simultaneously with caspase 3, CD10, activated b-catenin and survivin and the change in expression of activated b-catenin and survivin from diagnosis to relapse was measured by multiparameter phosphoflow cytometry in each patient by gating on the caspase 3 negative, CD10 positive leukemic blasts. To test the effect of Wnt inhibition on chemosensitivity, B-ALL cell lines were pretreated with iCRT14 for 48 hours prior to incubation with traditional chemotherapy for an additional 24 hours. The response to increasing doses of iCRT14 and chemo, alone and in combination, was assessed by cell viability (Cell Titer-Glo Luminescent Assay (Promega)) and apoptosis (FACS analysis with AnnexinV-PE/7AAD staining (BD Bioscience)). Protein levels of apoptotic markers were assessed. Also, 4 newly diagnosed and 4 relapsed patient samples were treated ex vivo with iCRT14 (20 and 30 uM) and prednisolone, alone and in combination. Drug combination results were analyzed using the Calcusyn program which calculates a Combination Index (CI): CI>1.1=antagonism, 0.9-1.1=additive and <0.9=synergy. Results Previously, we reported that treatment of ALL cell lines with iCRT14, downregulated the mRNA expression of the Wnt target genes BIRC5, axin 2, and c-myc (Romanos et al, ASPHO 2012 # 414). Comparison of Mean Fluorescent Intensity of activated b-catenin and survivin in the 10 pairs showed upregulation of activated b-catenin at relapse in 6 of 10 patients. Survivin expression was increased in 6 patients and in 4 patients the upregulation of activated b-catenin and survivin was concordant. iCRT14 pretreatment of cell lines followed by chemotherapy (prednisolone, etoposide, doxorubicin, cytarabine and 6TG) demonstrated additive to synergistic effects on viability. UOCB1 cells showed synergism with all 5 chemotherapy agents (CI=0.1-0.88). Nalm6 cells were very sensitive to iCRT, hence the combination with chemotherapy showed additive to synergistic effects (CI=0.05-1.I). In Reh cells, all agents besides cytarabine showed robust synergism (CI=0.03-0.55). FACS analysis revealed that iCRT14 alone contributed significantly to apoptosis and combination with chemotherapy further increased cell death with >80% apoptosis by hour 72 with the maximal chemotherapy dose in all cell lines. Change in the protein levels of cleaved PARP and cleaved caspase 3 was seen. The 4 diagnosis patients were very sensitive to prednisolone as expected, precluding synergism with iCRT14. The relapsed patient samples were much less sensitive to prednisolone alone (40% decrease in viability in relapsed patients vs 80% in new diagnoses). Interestingly, all the relapsed patients showed enhanced chemosensitivity with Wnt inhibition. 3 out of 4 relapsed patients showed strong synergism (CI=0.03-0.6) with both doses of iCRT14 and 1 patient showed additive to synergistic effects (CI=0.7 and 1). Conclusion Overactivation of the Wnt pathway may lead to chemoresistance in relapsed ALL. Wnt Inhibition restores chemosensitivity and induces apoptosis in ALL cell lines and primary patient samples making it a potential therapeutic approach. Disclosures: No relevant conflicts of interest to declare.

Transcription Activator-Like Effector Nucleases (TALENs)-Mediated Deletion Of MIR17HG In Burkitt Lymphoma Cells Decreases mTOR Pathway Activity and Increases Chemosensitivity

Background Burkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma in children. Although curable in most cases, those patients who relapse have a dismal prognosis with a 4-year survival of 16% (Cairo, Blood 2007). Identification of biomarkers to predict relapse/refractory disease is needed, and an understanding of the underlying mechanisms is vital to develop novel targeted therapies. We previously identified a recurrent gain of chromosome 13q31 that was associated with relapse and increased expression of miR-17-92 in pediatric BL (Schiffman/Miles, Br J Haematol, 2011). In the present study, we sought to determine the role of miR-17-92 expression in vitro using TALENs mediated deletion of the miR-17-92 locus (MIR17HG) in Raji BL cells that express high levels of miR-17-92 (Shiffman/Miles, 2011). Methods The modified restriction enzyme and ligation assembly method (Lee/Cairo, ASH 2012) was used to construct MIR17HG TALENs. Expression of miR-17 was assayed by TaqMan assay, and expression of the miR-17-92 target PTEN mRNA and protein were assayed by RT-PCR and western blot, respectively. Proliferation and caspase activity were measured at baseline and after treatment with cyclophosphamide. AKT and phospho (p)AKT were assayed by western blotting. To evaluate mTOR and MAPK pathway activation, pS6 and pERK were assayed by phosphoflow cytometry. In addition, caspase 3 activity was assayed by phosphoflow cytometry to assess induction of apoptosis. Cells were treated with chemotherapy (cyclophosphamide or 4-hydroperoxycyclophosphamide, 4HC), rapamycin, the PI3 kinase inhibitor PIK90, and the dual PI3K/mTOR inhibitor BEZ235 (Axon Medchem). To further validate the role of miR-17 in chemoresistance, miR-17 expression levels were measured by RT-PCR TaqMan assay in the previously developed rituximab-chemotherapy resistant Raji cell lines (RCRCL) Raji 2R and Raji 4RH (Barth et al. Br J Haematol, 2012). Results MIR17HG TALENs were used to generate two Raji cell lines with a hemizygous deletion of MIR17HG, #31(+/-) and #48(+/-). Relative to vector control Raji cells, cell lines with hemizygous MIR17HG deletion showed lower expression of miR-17: Raji = 1.0, #31= 0.80-fold, and #48= 0.71-fold. PTEN mRNA was higher in #31 and #48 cells than in the control (Raji = 1.0, #31 = 1.38-fold, and #48 = 2.24-fold), but there was no apparent difference in PTEN protein by western blot. Western blotting showed decreased pAKT with no difference in total AKT. Cyclophosphamide (10mM) induced a significant decrease in proliferation at 48 hours (90.5 +/- 2.9%, p=0.007) in #31 cells vs. control cells that was associated with a trend toward increased caspase 3/7 activity (148.3+/-8.9%, p=0.08). By phosphoflow cytometry, Raji #48 cells showed decreased pS6 compared to control cells and a modestly greater induction of caspase 3 in response to 4HC (3.4 uM) or PIK90 after 48 hours. The combination of 4HC and PIK90, however, significantly induced caspase 3 in Raji #48 cells relative to control Raji cells. Raji cells showed high basal levels of pERK and pS6. BEZ235 or PIK90 treatment decreased p-ERK. Rapamycin did not affect p-ERK but did decrease pS6. While BEZ235 and 4HC each modestly induced caspase alone in Raji cells, the combination caused a much greater increase in caspase 3. In RCRCLs Raji 2R and Raji 4RH, which show high pAKT levels by western blot and phosphoflow cytometry, miR-17 levels were increased (1.73- and 1.69-fold, respectively) relative to chemosensitive Raji cells (1.0) that exhibit lower pAKT levels. Conclusions Increased miR-17-92 expression in BL may contribute to therapy resistance, which is further supported by finding increased miR-17 expression in chemotherapy resistant Raji cell lines. Hemizygous deletion of the miR-17-92 locus in BL cells led to increased PTEN mRNA, decreased mTOR pathway activation (decreased pAKT and pS6), and increased sensitivity to chemotherapy. Resistance to PIK90 and 4HC in parental Raji cells that overexpresses miR-17-92 may be due in part to a compensatory upregulation of the MAPK and mTOR pathways, as demonstrated by increased pERK and pS6. Inhibiting this survival mechanism with the PI3K/mTOR inhibitor BEZ235 restored sensitivity to cytotoxic chemotherapy. These findings suggest potential mechanisms underlying BL therapy resistance as well as targeted therapies to overcome resistance. Disclosures: Cairo: Roche/Genentech: advisory board Other.

The Order of Exposure to Cytotoxic Chemotherapy and the Proteasome Inhibitor Bortezomib Dictates the Extent of Reversal of Chemotherapy Resistance in T-ALL

Abstract 1355 Introduction: While most children with T-cell acute lymphoblastic leukemia (T-ALL) are cured, the outcome for children who respond poorly to therapy or relapse is almost universally fatal. Our laboratory is interested in understanding the mechanisms contributing to therapy resistance and has developed a mutliparameter phosphoflow cytometry assay to study signal transduction events at a single cell level. Nuclear Factor kappa B (NF-κB) is a family of transcription factors that plays an important role in cancer development by preventing apoptosis and facilitating tumor cell growth. Consistent with this, constitutive NF-κB activity has been reported in T-ALL, and has been suggested as a predictor of relapse. We hypothesized that the proteasome inhibitor Bortezomib, which blocks NF-κB activation, induces apoptosis in T-ALL. We further hypothesized that combining Bortezomib with conventional chemotherapeutic agents sensitizes T-ALL cells to apoptosis and overcomes chemotherapy resistance by altering the balance of downstream pro- and anti- apoptotic mediators. Methods: A panel of T-ALL cell lines was cultured in the presence of vehicle alone, conventional cytotoxic drugs (Dexmethasone, Etoposide, Cyclophosphamide, Cytarabine), Bortezomib, or combinations of these. Cytotoxic effect was measured by trypan blue staining and flow cytometric detection of cleaved caspase-3. Multiparameter phosphoflow cytometry was used to measure expression levels of downstream apoptosis mediators. An advantage of multiparameter phosphoflow cytometry is the ability to gate on caspase 3-negative (i.e., chemotherapy resistant) cells, allowing us to interrogate potential mechanisms of resistance by comparing expression profiles of cell survival proteins before and after treatment. Results: T-ALL cells were exposed to cytotoxic chemotherapy or vehicle for 24 to 48 hours and subjected to phosphoflow cytometry. Analysis of caspase-3 negative cells indicated that chemotherapy resistant cells rewired their signaling networks by upregulating their MAPK, NF-κB, and AKT/PI3K/S6 signaling networks in response to genotoxic stress. This correlated with upregulation of the pro-survival protein survivin, suggesting a potential mechanism for chemotherapy resistance. To test whether Bortezomib could reverse this resistance, cells were exposed to subtoxic concentrations of Bortezomib alone or in combination with cytotoxic drugs. Combination therapy produced only a modest synergistic effect. We then wondered whether the sequence of administration of cytotoxic drug and Bortezomib could potentiate this effect. T-ALL cell lines were therefore 1) pretreated with Bortezomib for 24 hours followed by cytotoxic drug for an additional 24 hours, 2) pretreated with cytotoxic drug for 24 hours followed by addition of Bortezomib for an additional 24 hours, or 3) treated with both agents simultaneously. Surprisingly, the most pronounced cytotoxic synergy was observed when T-ALL cells were pretreated with cytotoxic drug followed by Bortezomib. While simultaneous or pretreatment with Bortezomib had no effect on upregulation of surivin in response to cytoxic chemotherapy, addition of Bortezomib post genotoxic stress resulted in markedly decreased levels of survivin. Conclusions: We found that Bortezomib lowered the apoptotic threshold to conventional cytotoxic drugs and can reverse drug resistance in T-ALL cell lines. Interestingly, the order of exposure to cytotoxic chemotherapy and Bortezomib determines the extent of reversal of chemotherapy resistance. Our data is in agreement with studies in multiple myeloma and provides evidence supporting the use of Bortezomib in combination with conventional cytotoxic agents in the treatment of relapsed or refractory disease. Disclosures: No relevant conflicts of interest to declare.

Thymic Stromal Lymphopoietin Stimulation of Pediatric Acute Lymphoblastic Leukemias with CRLF2 Alterations Induces JAK/STAT and PI3K Phosphosignaling

Abstract 410 Collaborative genomic profiling efforts though the National Cancer Institute's TARGET Initiative and the Children's Oncology Group have identified CRLF2 and JAK mutations in a subset of children with high-risk acute lymphoblastic leukemia (ALL), but few biochemical studies have assessed the functional sequelae of these genetic alterations. CRLF2 encodes the thymic stromal lymphopoietin (TSLP) receptor chain, which heterodimerizes with the IL-7 receptor alpha chain (IL-7Rα). Children with high CRLF2-expressing ALL detected by gene expression profiling have high rates of minimal residual disease at end-induction (Day 29), and approximately 70% of these patients ultimately relapse (Harvey et al., Blood 2010). We hypothesize that characterization of aberrant signaling networks in these leukemias will facilitate identification of potential targets for small molecule inhibitor therapies. Using phosphoflow cytometry, we analyzed the phosphorylation status of key signaling molecules after stimulation with TSLP, IL-7, or pervanadate (an irreversible proximal membrane phosphatase inhibitor used as a positive control) in 2 human ALL cell lines with CRLF2 and JAK2 mutations and in 43 fresh or cryopreserved diagnostic primary patient samples, 27 of which overexpressed CRLF2 through P2RY8-CRLF2 fusion or CRLF2-IgH translocation and 16 of which did not have CRLF2 or JAK mutations (controls). Cells were rested in serum-free media for 60 minutes at 37°C, then stimulated with TSLP, IL-7, or pervanadate for 30 minutes to induce signaling. Cells were also exposed to the JAK inhibitor XL019 (Exelixis) for 60 minutes and/or subsequently stimulated with the aforementioned cytokines or pervanadate to determine the effects of JAK inhibition on signaling. Cells were then processed for phosphoflow cytometry according to our previously published methodologies (Kotecha et al., Cancer Cell 2008). High CRLF2-expressing leukemias (n = 27) with or without concomitant JAK mutations demonstrated strong surface staining of the TSLP receptor, as well as CD10, CD19, and CD127 (IL-7Rα). In vitro stimulation of leukemic blasts with TSLP elicited phosphorylation of STAT5 and S6, but not ERK 1/2, in leukemias with JAK and/or CRLF2 alterations. Control leukemias without CRLF2 and JAK mutations (n=16) did not stain for the TSLP receptor, and TSLP stimulation did not elicit phosphosignaling through the JAK/STAT, PI3K, or MAPK pathways. STAT5 and S6 phosphorylation in the high CRLF2-expressing leukemias was further abrogated by in vitro JAK inhibition with XL019. Surprisingly, despite flow cytometric staining for CD127, stimulation with IL-7 did not elicit phosphosignaling through these epitopes in high CRLF2-expressing or control leukemic blasts, although it did predictably phosphorylate STAT5 in control T and non-blast B cells contained within the primary patient leukemia samples. These results suggest that the JAK/STAT and PI3K pathways, but not the MAPK pathway, are involved in TSLP receptor signaling in high CRLF2-expressing ALL +/− JAK mutations and may represent druggable targets. Phosphoflow cytometry is an efficient method of interrogating intracellular signaling at a single-cell level in primary human samples and, furthermore, can be used for rapid identification of patients at time of leukemia diagnosis with high CRLF2-expressing ALL who exhibit the TSLP phosphosignature. Therapy for this subset of high-risk patients might be modified to include a targeted therapeutic (such as a JAK inhibitor) to improve initial treatment responses and, ultimately, to enhance long-term survival. To this end, we have developed a Children's Oncology Group Phase I clinical trial of JAK inhibition for patients with relapsed or refractory leukemias (including those with CRLF2 and JAK mutations) and will validate the use of phosphoflow cytometry and other biologic assays to assess in vivo target inhibition during therapy. We ultimately envision incorporation of JAK inhibitor therapy into a systemic chemotherapy backbone for patients with high CRLF2-expressing ALL. Disclosures: No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) Signaling Profiling Identifies CLL-Specific Signaling Impairment That Discriminates CLL B-Cells From Normal Circulating B Cells

Abstract 1374 CLL is the most common leukemia in the Western world. It is characterized by the clonal expansion of CD5+/CD19+ B cells, expressing surface immunoglobulins (sIg), most often of the IgM subset. Clinically, it can either manifest as an indolent disease with little impact on the lifespan of a subset of patients or as an aggressive, highly drug-resistant disease with lethal outcome but all possible scenarios in between these extremes are routinely observed. The degree of somatic mutations of the sIg has been shown to affect survival; patients expressing highly mutated sIg have a better prognosis than patients whose sIg are less than 2% different from germline sequences. Therefore, it is intuitive that signaling through the sIg, or B-cell receptor (BCR), must have a role in determining the fate of the CLL cells, veering the intracellular machinery towards proliferation or apoptosis. We have systematically investigated the downstream events caused by BCR stimulation via crosslinking of the sIg in a series of about 60 heterogeneous CLL patients and 15 normal donor peripheral blood mononuclear cells (PBMCs). Using multiplexed phosphoflow cytometry we were able to assess the intensity of multiple signaling events at specific stages of the BCR signaling pathway. By combining phosphospecific staining and classic immunophenotyping we were also able to evaluate signaling intensity at the at the single cell level as well as correlating the signal intensity of CLL-specific surface markers to that of the phosphoproteins analyzed. We used principal component analysis to combine phosphospecific staining within individual cells and generate a variable that discriminates efficiently between CLL and PBMCs B-cells. We found that signaling properties of CLL B-cells were impaired when compared to normal B cells from healthy donors, both at the level of upstream members of the BCR signaling pathway (pBLNK, pSYK, pZap70, pBTK, pPLCg2) and downstream components such as pp38 and pERK. However, within the cohort of CLL samples we identified a small subpopulation in which signaling proceeded as well or more efficiently than in normal B cells. We also used our principal component variable to quantify the heterogeneity of signaling responses within populations of CLL B-cells and correlate them with clinical parameters that are known prognostic indicators in CLL. In conclusion, multiplexed phosphoflow cytometry of individual CLL cells provides a novel and highly specific method to determine the use of signaling pathways (such as BCR-mediated) in comparison to normal B cells. Our studies indicate a significant decrease in BCR signaling of CLL B cells, which involves both upstream and downstream components. Disclosures: van den Brink: Cytheris: Research Funding.