ALBERT

Description: Key Points In B-ALL, cells that express a functional pre-BCR ibrutinib abrogate leukemia cell growth in vitro and in vivo. Effects of ibrutinib in B-ALL not only are mediated through inhibition of BTK but also involve BLK inhibition.

Description: Abstract 2569 Bruton's tyrosine kinase (BTK) is a member of the TEC family of non-receptor tyrosine kinases and is predominantly expressed in hematopoietic cells, except in T cells. BTK plays a prominent role in B cell receptor (BCR) signaling and several other pathways, including CXCR4 signaling, which is essential for lymphocyte homing. BTK activation downstream of the BCR leads to proliferation, differentiation, and survival of B cells. Functional BTK is necessary for normal B cell development, defective BTK result in a primary immunodeficiency called X-linked agammaglobulinemia (XLA). Because of the restricted expression and the B cell phenotype in BTK-deficient mice and XLA patients, BTK has become a promising therapeutic target in mature B cell malignancies. Ibrutinib is a selective, orally bioavailable, covalent BTK inhibitor currently studied in clinical trials in patients with Chronic Lymphocytic Leukemia (CLL) and other mature B cell malignancies. The importance of BTK in B-ALL is controversial. Initial study in childhood B-ALL revealed normal BTK protein levels, and gene expression profiling data from the St. Jude's group revealed BTK expression in B-ALL, but not in T-ALL. Other studies revealed abnormally spliced BTK mRNA and truncated BTK protein lacking kinase activity in some B-ALL samples. To explore the pre-clinical activity of ibrutinib in B-ALL, we used a panel of 14 B-ALL cell lines, representing pro-B (CD10+/−, TdT+, cyt Igμ-), pre-B (CD10+, TdT+, cyt Igμ+) and mature (CD10+/−, TdT-, surf IgM+) phenotypes. The panel included 4 Ph+/BCR-ABL1+ cell lines (Z-119, NALM-20, NALM-21, TOM-1). Western blot analysis revealed BTK expression in 12 out of 14 lines, while phospho-BTK (Y223) was present only in half of the cases. Effects of ibrutinib on B-ALL proliferation were tested in XTT assays, using increasing concentrations of ibrutinib (0.5 – 5 μM). All B-ALL cells responded to ibrutinib except for BTK-negative TANOUE cells. All other B-ALL cells displayed decreased proliferation with variable half maximal inhibitory concentrations of ibrutinib (IC50). The most sensitive cell lines (RCH-ACV, SMS-SB) had IC50 of 〈 1 μM; all BCR-ABL1+ cells showed IC50 〈 3.5 μM (see Figure). We also analyzed inhibitory effects of ibrutinib on B-ALL cell proliferation by serial automated cell counting, which confirmed the XTT assay data. B-ALL cells viability was determined after incubation with ibrutinib, which induced only minor decreases after 3 days of incubation. Preliminary data with primary B-ALL samples revealed BTK protein expression in 5 out of 5 samples. In co-culture with KUSA-H1 stromal cells, primary B-ALL cells showed moderate levels of apoptosis, ranging from 10 – 25% after 3 days of incubation with 1 μM ibrutinib, which is similar to levels of ibrutinib-induced apoptosis in CLL. Collectively, these data demonstrate that the BTK inhibitor ibrutinib can interfere with B-ALL cell proliferation and survival, providing a rationale for clinical testing of this novel, well-tolerated targeted agent in patients with relapsed B-ALL. Disclosures: O'Brien: Pharmacyclics: Research support Other. Buggy:Pharmacyclics: Employment, Equity Ownership. Burger:Pharmacyclics: Consultancy, Research Funding.

Description: B lymphocyte development proceeds in a stepwise fashion and is tightly linked to the generation of a functional B cell receptor (BCR). At the preB cell stage B lymphocyte progenitors express the precursor B cell receptor (pre-BCR), an immature form of the BCR consisting of two µ heavy chains (µHC) and two surrogate light chains (SLC). Pre-BCR expression marks the proB to preB transition and induces a burst in preB lymphocyte proliferation. In 20% of the cases B cell acute lymphoblastic leukemia (B-ALL) arises from lymphocytes arrested at the pre-BCR positive stage of lymphocyte development (preB-ALL). Due to the essential role of the pre-BCR for preB cell proliferation we hypothesized that pre-BCR signaling also is involved in the maintenance of preB-ALL. Consequently, pharmacological inhibition of Spleen tyrosine kinase (Syk), the main transducer of pre-BCR signaling, may serve as effective treatment for this subtype of B-ALL. We analyzed a panel of six ALL cell lines (SMS-SB, RCH-ACV, Nalm-6, Kasumi-2, 697, KOPN-8) arrested at the pre-BCR+ stage of B lymphocyte development (cytoIgµ+, sIgM-). Assessment of the baseline phosphorylation levels of the pre-BCR associated kinases Lyn, Syk and Btk by immunoblotting and subsequent densitometric analysis allowed us to assign B-ALL cells into groups with either high levels of Lyn, Syk and Btk phosphorylation or with low or absent phosphorylation of these kinases, respectively. Moreover cell lines with highly phosphorylated Lyn, Syk and Btk also exhibited lower surface pre-BCR expression than cell lines with low phosphorylation levels. As pre-BCR activation is followed by its rapid internalization the concomitant presence of low pre-BCR expression and high phosphorylation of pre-BCR associated proteins suggests increased pre-BCR pathway activity. When we investigated the impact of pharmacological inhibition of the pre-BCR associated kinase Syk through the highly specific inhibitor PRT060318, preB-ALL cell lines with highly phosphorylated pre-BCR associated molecules turned out to be more sensitive to Syk inhibition (IC50 〈 1.6µM) than preB-ALL cell lines with less phosphorylation (IC50 〉 3.9µM). In proliferation assays PRT060318 inhibited preB-ALL proliferation in a dose dependent manner, whereas PRT060318 did not induce apoptosis in concentrations as high as 5µM. This supports the notion that pre-BCR signaling activity may be more relevant for preB-ALL proliferation than for preB-ALL viability. In line with these results the pre-BCR- proB-ALL cell lines REH and RS4;11 were highly resistant to Syk inhibition in all functional assays (IC50 〉 10µM), suggesting that pre-BCR expression is a prerequisite for sensitivity to Syk inhibition. To examine the molecular changes following pre-BCR inhibition, ALL cells were treated with increasing concentrations of PRT060318 (100nM-5µM) for two hours and then subjected to immunoblotting. Syk inhibition led to a dose dependent decrease in AKT phosphorylation in all preB-ALL cell lines and subsequently reduced phosphorylation of FOXO transcription factors. In the resistant proB-ALL cell line REH, AKT and FOXO phosphorylation were not affected. Gene expression analysis of the preB-ALL cell lines RCH-ACV and Nalm-6 further suggested that PRT060318 interferes with pre-BCR signaling. Treatment with 1µM PRT060318 for 72h reduced the expression of genes associated with pre-BCR signaling (e.g. BCL6, CD22, PTPN6) and Ingenuity Pathway Analysis identified pre-BCR signaling as the main target of PRT060318 in both cell lines (p

Description: Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL? Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells. Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells. In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2). Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement. Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Disclosures Westin: Spectrum: Research Funding.

Description: Deregulated cell death and survival pathways contribute to leukemogenesis and treatment failure of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. The intrinsic apoptosis pathway is regulated at the mitochondrial level by different pro- and anti-apoptotic molecules. Members of the BCL-2 family are key regulators of mitochondrial apoptosis signaling. Pro-apoptotic BH3-only proteins like BIM and BID activate pro-death proteins such as BAX and BAK leading to cell death. Anti-apoptotic BCL-2 family members including BCL-2, BCL-XL and MCL-1 bind to and sequester pro-apoptotic molecules, prevent activation of pro-death proteins and counter-regulate apoptosis induction. Small molecule inhibitors have been developed that block binding to anti-apoptotic molecules like BCL-2, leading to release of pro-apoptotic proteins and cell death induction. In particular, the BCL-2-specific inhibitor venetoclax (VEN) has demonstrated substantial anti-cancer activity and became an approved drug for the treatment of CLL patients. Investigating different, individual BCP-ALL samples, we and others recently identified heterogeneous sensitivities for VEN, suggesting that BCP-ALL cells might also depend on other pro-survival BCL-2 family proteins including MCL-1, leading to VEN insensitivity and resistance. A novel BH3-mimetic, S63845, that selectively targets MCL-1 has been reported. Here, we assessed the activity of S63845 and addressed a potential synergism of simultaneous blockage of BCL-2 and MCL-1 by VEN and S63845 (S) in BCP-ALL. The activity of the MCL-1 inhibitor was analyzed in a panel of BCP-ALL cell lines (N=6) and a series of primary, patient-derived BCP-ALL primograft samples (N=27) determining half-maximal effective concentrations (EC50) upon exposure to increasing concentrations of S and analysis of cell death induction. We observed heterogeneous sensitivities to S with EC50 values ranging from 16 nM to almost 10 µM. Protein expression of MCL-1 and other BCL-2 family members BCL-2, BCL-XL and BCL-W was assessed by western blot analysis and quantified, however neither association of MCL-1 levels nor expression of the other regulators and S sensitivity was found in cell lines and primograft leukemias. Moreover, we also compared sensitivities for both inhibitors but found independent activities of S and VEN in individual ALL samples. Next, we addressed the role of MCL-1 for VEN sensitivity and generated two MCL-1 knock out BCP-ALL cell lines by CRISPR/Cas9 gene editing. In both lines, clearly increased VEN sensitivities were observed upon depletion of MCL-1, indicating that MCL-1 is contributing to activity of the BCL-2 inhibitor VEN. Based on these findings, we investigated the effects of pharmacological MCL-1 inhibition for VEN sensitivity and incubated all 6 cell lines with VEN and S at increasing concentrations and observed clear synergistic effects upon combined BCL-2 and MCL-1 inhibition indicated by combination indices (CI) below 0.1. Moreover, we investigated 7 primograft BCP-ALL samples and found that MCL-1 inhibition by S clearly synergized with VEN activity (CI 〈 0.3). To investigate the anti-leukemia activity of co-targeting BCL-2 and MCL-1 in vivo in a pre-clinical setting, a high-risk leukemia derived from an infant, MLL/ENL rearranged pro-B ALL case was transplanted onto NOD/SCID mice. Upon ALL manifestation (presence of 〉5% human blasts in blood), recipients were treated with either VEN, S, the combination of both, or vehicle for 10 days. After treatment, leukemia loads were analyzed showing significantly reduced loads in the co-treated group as compared to vehicle, VEN or S alone in spleen, bone marrow, and central nervous system (p-values 〈 0.05), indicating synergistic activity of co-inhibition of BCL-2 and MCL-1 in vivo. Taken together, our data show heterogeneous sensitivity of individual BCP-ALL samples to MCL-1 inhibition by S, which is not associated with MCL-1 protein expression levels or VEN sensitivity. Both, genetic depletion and inhibition of MCL-1 by S synergizes with VEN leading to increased anti-leukemia activity in vitro and ex vivo. Importantly, co-targeting BCL-2 and MCL-1 significantly reduced leukemia infiltration in spleen, BM and CNS in a pre-clinical model of high-risk BCP-ALL, warranting further evaluation and possible clinical application of targeting MCL-1 alone and in combination with BCL-2 inhibition. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Targeting antigen-driven B-cell receptor (BCR) signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), but not germinal center B-cell (GCB) DLBCL. We have formally confirmed that GCB-DLBCL cell lines utilize tonic BCR signaling, by showing: 1) sensitivity (variable) to knockout (KO) of the BCR, SYK, and CD19; 2) dependence on CD79A ITAM phosphorylation; and 3) independence from BCR antigen specificity. However, uncertainty remains about molecular events in upstream parts of tonic BCR signaling, why dependence of GCB-DLBCL cells on tonic BCR signaling is variable, and their clinical relevance. Methods. We used CRISPR/Cas9 methods to modify selected genes by KO and/or knock-in (KI) of the cDNA of a fluorescent protein (FP; e.g., GFP), with the FP serving as a marker of cells with gene KO or modification, or as a gene-fused tag for localization or quantitation. Cells expressing a membrane-targeted Forster resonance energy transfer (FRET) based AKT activity reporter (Lyn-AktAR2) were used to measure AKT activity directly by flow cytometry (FCM). Results. The effect of KI of CD79A Y188F mutation alone was similar to complete BCR KO, implying that CD79A Y188 phosphorylation is essential for tonic BCR signal transduction. Western blot analysis of GCB-DLBCL cell lines after BCR KO showed variable decreases of AKT S473 phosphorylation (frequently used as surrogate measure of AKT activity), but these did not correlate well with the variable decreases in proliferation of GCB-DLBCL cell lines caused by BCR KO. Measuring AKT activity directly (Fig. 1), or by another indirect approach (surface expression of CXCR4, a target gene of FOXO1 inhibited by AKT activity), showed high correlation between decreases in AKT activity and proliferation after BCR KO. In contrast to the variable effect of BCR KO on growth, pan-AKT KO was uniformly growth-slowing in GCB-DLBCL lines (Fig. 2). Interestingly, baseline surface density of BCR units in GCB lines, quantified by FCM using CD79A-GFP KI cells or anti-CD79B staining, correlated highly with reduction in growth or AKT activity caused by BCR KO (Fig. 3). These findings lead us to conclude that the BCR contributes to AKT activation in GCB-DLBCL cell lines, to a variable degree determined by BCR surface density. We also conclude that BCR surface density is determined by cell line-specific factors, as well as immunoglobulin heavy (IgH) and light (IgL) hypervariable region (HVR) sequences, based on measurements of BCR surface levels after exchanging endogenous HVR sequences in OCI-Ly19 and OCI-Ly7 cell lines for HVRs derived from other GCB and ABC-DLBCL cell lines. Reduction of AKT activity after BCR KO (measured by FRET reporter) and baseline BCR surface density in GCB-DLBCL cell lines also correlated well with the sensitivity of GCB-DLBCL lines to the clinically-tested SYK inhibitor (P505-15, PRT062607) or FDA-approved PI3K p110d isoform specific inhibitor (idelalisib). Interestingly, isogenic GCB-DLBCL cell lines with KO of PTEN, a negative regulator of AKT activation, were substantially more resistant to both inhibitors. A crucial role of PTEN deletion in overcoming dependence on tonic BCR signaling in GCB-DLBCL is supported by evidence from two naturally PTEN-deficient cell lines: SUDHL10, which adjusts to BCR KO and resumes normal growth, and HT, which lacks BCR expression, due to a frameshifting deletion in its IgH HVR. Re-expression of the BCR in HT, by KI to correct the IgH sequence, does not affect HT cell line growth. Conclusion. Our findings suggest a biomarker-guided therapeutic strategy in GCB-DLBCL: targeting tonic BCR signaling in BCR-high patients, by inhibiting CD79A phosphorylation, SYK, or PI3K, and downstream targeting of AKT in BCR-low and/or PTEN-deficient patients. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 1. Correlation of relative proliferation after BCR KO with decrease of AKT activity (as measured by FRET efficiency of AKT activity reporter) in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 2. Effect of BCR KO or pan-AKT KO in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Figure 3. Correlation of relative proliferation after BCR KO with baseline BCR surface density (as measured by flow cytometry of cells with CD79A-GFP fusion) in GCB-DLBCL cell lines. Disclosures Burger: Pharmacyclics: Research Funding. Westin:Chugai: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; ProNAi: Membership on an entity's Board of Directors or advisory committees.

Description: Despite superior outcome and survival of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), relapse occurs in 10-20% and is associated with poor outcome, clearly indicating future challenges including reduction of relapse rates and effective treatment of reoccurred leukemia. Deficiencies in cell death and survival pathways have been implicated in therapy failure and treatment resistance in BCP-ALL. Members of the BCL-2 family are key regulators of these pathways and are therefore of interest as therapeutic targets. The small molecule ABT-199 binds selectively to BCL-2, inhibits its anti-apoptotic function and leads to release of pro-apoptotic molecules. Recently, ABT-199 has demonstrated clinical activity, particularly in poor prognosis CLL. However, insensitivity and resistance in different cases clearly emphasize the need of predictive markers for upfront identification of ABT-199 responsive leukemias. Here, we analyzed sensitivity for ABT-199 in a series of individual BCP-ALL samples, addressed mechanisms of resistance and evaluated markers indicating response to ABT-199. Anti-leukemic activities of ABT-199 were investigated in BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primograft samples (n=17), which were established by transplantation of primary patient ALL cells obtained at diagnosis onto NOD/SCID mice. Half maximal inhibitory concentrations (IC50) for ABT-199 were analyzed for each sample. Expression of apoptosis regulating molecules was investigated by western blot analysis and associated with ABT-199 responsiveness. Two MCL-1 deficient ALL cell lines were generated by CRISPR/Cas9 gene editing. Leukemia free-survival of ALL bearing animals was analyzed after in vivoABT-199 treatment. The majority of BCP-ALL samples showed sensitivity for ABT-199 induced cell death in the nanomolar range, both in cell lines (n=4, IC50: 29 - 422 nM) and patient-derived primograft samples (n=10, IC50: 1.7 - 74 nM), while 2 cell lines and 7 primograft leukemias showed insensitivity with IC50 values above 1 µM. ABT-199 binds directly to BCL-2 and upon binding, pro-apoptotic Bcl-2 family molecules like Bim are dislocated from BCL-2 and induce apoptosis. The anti-apoptotic BCL-2 family member MCL-1 is not bound by ABT-199, but sequesters pro-apoptotic molecules dislocated from BCL-2 leading to interruption of apoptosis induction. Therefore, we addressed expression levels of BCL-2 and MCL-1. We found high BCL-2 levels in ABT-199 sensitive and low BCL-2 levels in resistant leukemia samples and an opposite pattern for MCL-1 (high in resistant and low MCL-1 in sensitive ALL), in line with previous reports. Most interestingly, a high ratio of MCL-1 to BCL-2 expression (high MCL-1, low BCL-2) was significantly associated with high IC50 values/resistance (Spearman Rho correlation, p= .01), whereas a low MCL-1/BCL-2 ratio indicated ABT-199 sensitivity. Two of the 6 cell lines showed ABT-199 resistance (IC50 〉 1 µM) and high Mcl-1 expression. Effective MCL-1 knock-out in both cell lines led to a clear sensitization for ABT-199 with up to 40-fold reduced IC50 values, clearly indicating MCL-1 as a key mediator of ABT-199 resistance in BCP-ALL. Finally, we also evaluated the anti-leukemia activity of ABT-199 in a preclinical setting in vivo. Two patient-derived leukemias, one with a low MCL-1/BCL-2 ratio of 0.9 and the other with a high ratio of 16.1, indicative of ABT-199 sensitivity or resistance, were transplanted onto NOD/SCID mice and treated with ABT-199 for 10 days after ALL engraftment. Most interestingly, a significantly increased leukemia free survival was observed in ABT-199 as compared to vehicle treated recipients (p

Description: Introduction. An essential role for the B-cell receptor (BCR) has been shown in multiple types of B-cell lymphoma by studies of cell lines and clinical responses to inhibitors of SYK or BTK. Diffuse large B-cell lymphoma (DLBCL) lines of the germinal center B-cell (GCB) type express a BCR, which can signal after crosslinking, but are unaffected by BCR pathway targeting toxic to lines of the activated B-cell (ABC) DLBCL subtype: knockdown of BCR signaling mediators (BTK, CD79A, and CD79B) by shRNA, and small-molecule inhibition of BTK by ibrutinib. GCB-DLBCL lines (and primary samples) also lack constitutive NF-kB activity and mutations in ITAM domains of CD79A or CD79B, BCR-related features of ABC-DLBCL. Most GCB-DLBCL patients resist BTK inhibition by ibrutinib, further suggesting that BCR signaling is not a feature of GCB-DLBCL. Methods. In 8 GCB-DLBCL lines (OCI-Ly7, OCI-Ly19, SUDHL-4, SUDHL-6, SUDHL-10, DB, BJAB, and HT) and one ABC-DLBCL line (HBL-1), we used electroporation to deliver a plasmid expressing Cas9 protein and a guide RNA (gRNA) targeting one of these: constant exons of IGHM, IGHG, or Igκ; the cell line-specific IgH hypervariable region (HVR); or CXCR4. Knock-in (KI) of mouse CD8a (mCD8a), after the HVR V segment leader sequence and followed by a polyA signal, was used as a positive marker of BCR knockout (KO) in HBL-1 and OCI-Ly19 cell lines. Surface BCR, CXCR4, and mCD8a were detected by flow cytometry (FACS). BCR KO cells were viably sorted 4-6 days after electroporation, cultured 1-3 days more, and studied by whole-genome gene expression profiling (GEP) on Illumina HT12v4 arrays and Western blotting. Results. Only 2 days after electroporation, FACS showed cells with correlated loss of surface BCR proteins (IgH, Igκ or Igl, and CD79B), which eventually declined to undetectable levels. Forward and side scatter showed that BCR KO cells were smaller. The proportion of BCR KO (or mCD8a KI/KO) cells declined over time, steadily after complete BCR elimination (Fig. 1A). BCR KO cells in GCB-DLBCL lines grew more slowly than BCR-replete cells but variably, from almost no difference in BJAB to growth cessation in SUDHL-4, SUDHL-10 and HBL-1 (Fig. 1B). CXCR4 KO cells were a stable proportion (Fig. 1A) with a normal growth rate (Fig. 1B), indicating that growth reduction by BCR KO is specific. Continued expression of mCD8a indicated viability and sustained IgH transcription in BCR KO cells. Cell cycle analysis showed lower proportions of S and G2/M phases in BCR KO cells, proportional to growth retardation, and sub-G1 cells in OCI-Ly7 (Fig. 2), SUDHL-4 and SUDHL-10. Apoptosis in OCI-Ly7 BCR KO cells was confirmed with a caspase-3 fluorogenic substrate. Igκ KO similarly caused complete BCR loss and growth retardation, in OCI-Ly7 cells even more than with IgH KO. In the HT cell line, which lacked BCR expression due to a single-nucleotide deletion in its IgH HVR, KI repaired the HVR and caused expression of surface BCR (IgM with Igκ and CD79B) but no change in growth rate, suggesting BCR-proximal activators of BCR signaling pathways. Targeted BCR KO is not currently a therapeutic option, but BCR KO cells were relatively more sensitive to an in vitro regimen modeling the non-prednisone drugs of CHOP. No change in drug sensitivity was observed with BCR KO in BJAB, or in CXCR4 KO cells. GEP showed that BCR KO downregulated several genes characteristically expressed by GCB-DLBCL, and genes associated with negative regulation of BCR signaling. Pathway analysis with Gene Set Enrichment Analysis (GSEA) showed that BCR KO reduced expression of proliferation-related signatures, and produced changes associated with B-cell differentiation stages lacking a mature BCR, either early (pre-B cells) or late (plasma cells). GSEA implicated loss of MAPK/ERK and PI3K/AKT signaling pathways as mediators of BCR KO-induced changes, confirmed by Western blotting showing loss of phosphorylation of SYK, AKT and ERK after BCR KO. Conclusions. Complete BCR KO by Cas9/gRNA showed that GCB-DLBCL lines require the BCR for optimal viability, cell growth, and chemotherapy resistance. BCR KO-induced changes are mediated by MAPK/ERK and PI3K/AKT signaling pathways. Table A. B. Figure 1. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1A. BCR KO cells (distinguished from BCR-replete cells by FACS), but not CXCR4 KO cells, show relative decline (A) and slower absolute growth (B) in mixed cultures. Figure 1B Figure 1B. Figure 2 Cell cycle changes with BCR KO in OCI-Ly7. Figure 2. Cell cycle changes with BCR KO in OCI-Ly7. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Bruton′s tyrosine kinase (BTK) is a member of TEC family of non-receptor tyrosine kinases. BTK is mostly expressed in hematopoietic cell lineages, except in T cells. It plays a particularly important role in B cell development and is present at almost all stages of their maturation, disappearing only in plasma cells. BTK is an essential kinase downstream of pre-B cell (pre-BCR) and B cell receptors (BCR) promoting proliferation, differentiation and survival of B cells. Methods Surface protein expression in B-cell acute lymphoblastic leukemia (B-ALL) cell lines was assessed by flow cytometry using PE conjugated anti-CD179a, anti-CD179b (BioLegend) and anti-CD22 (BD Pharmingen). To investigate effects of the BTK inhibitor ibrutinib (PCI-32765) on constitutive pre-BCR signaling RCH-ACV cells were pretreated with 0.1% DMSO or increasing concentrations of the drug (0.0001, 0.001, 0.01, 0.1, 1.0 μM) for 1 hour and lysed in RIPA buffer. To induce pre-BCR signaling in pretreated cells they were incubated with 10 μg/ml of anti-Igμ for 30 minutes. Intracellular calcium mobilization was measured by using the fluorogenic probe Fluo3-AM (Invitrogen). RCH-ACV cells pretreated with 1 μM ibrutinib for 72 hours were subjected to gene expression profile analysis on HT-12 v4 Expression BeadChip (Illumina). Results Previously we explored the effects of ibrutinib in B-ALL cell lines and primary samples. Ibrutinib induced only low levels of apoptosis in B-ALL cell lines, but significantly inhibited their proliferation. RCH-ACV and SMS-SB were the most sensitive cell lines with half maximal inhibitory concentrations of ibrutinib of 0.6 and 0.4 μM found in XTT cell proliferation assay. Interestingly, both cell lines expressed a pre-B cell immunophenotype with pre-BCR surface expression. Next, we explored the effect of BTK inhibition on constitutive and induced pre-BCR signaling. Treatment of RCH-ACV cells with varying concentrations of ibrutinib resulted in decreased levels of pBTK, pAKT, pS6 and pSYK. The lowest concentration of ibrutinib needed to observe complete disappearance of pBTK (Y223) and any reduction of other phospho-proteins was 10 nM, however the maximum effect was achieved with 1 μM ibrutinib. Upon pre-BCR crosslinking with anti-Igμ elevated levels of pSYK, pBTK, pAKT, pS6 and pERK were detected in RCH-ACV. Pretreatment of the cells with ibrutinib greatly reduced this effect. As calcium mobilization is another important indicator of B cell activation upon pre-BCR stimulation, we evaluated ibrutinib in calcium flux assays. Pretreatment with 1 μM ibrutinib effectively abrogated anti-Igμ induced calcium flux in pre-B ALL cell lines. Gene expression profile analysis of RCH-ACV cells after 72 hours of incubation with 1 μM ibrutinib showed down-regulation of pre-BCR related genes such as PTPN6 (SHP-1), Bcl6 and CD22. Flow cytometry analysis confirmed the down-regulation of the inhibitory co-receptor CD22 in pre-B ALL cell lines after incubation with ibrutinib. The down-regulation of SHP-1 protein was verified by western blotting. Conclusions The results indicate that ibrutinib reduces the pre-B ALL cell proliferation by inhibiting constitutive and/or induced pre-BCR signaling. Observed down-regulation of CD22 and SHP-1, known negative regulators of BCR signaling, suggests a possible mechanism of cell adaptation to the presence of the BTK inhibitor. Taken together, these data provide a rationale for clinical testing of ibrutinib in B-ALL with active pre-BCR signaling. Disclosures: O'Brien: Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment. Burger:Pharmacyclics: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Description: A pivotal step during B-cell development is the expression of the precursor B-cell receptor (pre-BCR) by pre-B lymphocytes (cyto-Igµ+, surface-IgM-). The pre-BCR represents an immature form of the BCR and consists of two immunoglobulin heavy chains (IgH), two surrogate light chains (SLC) and the signal transducing adapter proteins Igα and Igβ. A functional pre-BCR drives proliferation of pre-B-cells, ensuring their further differentiation into mature B-cells. By immunophenotype, ~20% of B-cell acute lymphoblastic leukemia (B-ALL) cases originate from the pre-B-cell stage (pre-B-ALL) of lymphocyte development and might therefore also express the pre-BCR. In view of the importance of pre-BCR signaling for normal pre-B-cell development, we hypothesize that it is exploited by pre-B-ALL for malignant growth and proliferation. A hallmark of active pre-BCR signaling is the continuous internalization of pre-BCRs, resulting in low pre-BCR surface expression. Using this phenotype of active pre-BCR signaling (low pre-BCR expression and high phosphorylation of the pre-BCR associated kinases LYN and SYK), we identified pre-BCR+ ALL cell lines (RCH-ACV, SMS-SB and Nalm6) and xenograft expanded patient samples. To study the role of the pre-BCR in these cells, we rendered RCH-ACV and SMS-SB pre-BCR null by using CRISPR/CAS9 gene editing with guide RNAs specific for the hypervariable region (recombined V, D, and J segments) of their expressed IgH allele. As identified by flow cytometry for the pre-BCR, deficient RCH-ACV and SMS-SB cells exhibited reduced viability and impaired proliferation when compared to their pre-BCR+ controls (Figure 1). Pre-BCR- cells showed reduced baseline phosphorylation of CD19, VAV1 and AKT. Interestingly, BTK and ERK phosphorylation were not affected. These results provide evidence for the dependency of pre-BCR+ ALL on pre-BCR signaling and suggest selective involvement of the PI3K-AKT pathway. We also investigated the effects of pharmacological pre-BCR inhibition by treating pre-BCR+ and pre-BCR- ALL cell lines and xenograft expanded primary patient samples with PRT318, a small-molecule inhibitor of spleen tyrosine kinase (SYK). In pre-BCR+ ALL PRT318 blocked cell proliferation and selectively inhibited AKT phosphorylation, thus mimicking the effects of IgH knockout. Pre-BCR- ALL cells were resistant to PRT318. Key effectors of the pre-BCR during normal B-cell development are FOXO transcription factors. In line with this, we found reduced FOXO1 phosphorylation and increased FOXO1 total protein levels after IgH knockout as well as after treatment with PRT318. This was accompanied by an increase in the FOXO1 transcriptional targets p27 and BLNK, suggesting increased FOXO1 transcriptional activity in response to the inhibition of pre-BCR signaling. To study the contribution of FOXO1 to the effects of IgH knockout and SYK inhibition more thoroughly, we expressed constitutively active FOXO1 (FOXO1-3A) in the pre-BCR+ ALL cell line RCH-ACV and consequently assessed its effects on cell proliferation and protein expression. Similar to IgH knockout and PRT318, FOXO1-3A reduced cell proliferation and increased p27 and BLNK protein levels, confirming FOXO1 as an important downstream target of pre-BCR signaling in B-ALL. To identify additional effectors of the pre-BCR in B-ALL we performed gene expression profiling (GEP) to compare pre-BCR+ and pre-BCR- cells of RCH-ACV and SMS-SB. Gene set enrichment analysis (GSEA) showed that IgH knockout resulted in significant enrichment for gene sets associated with down-modulation of MYC activity. This was confirmed by Western blot analysis of MYC total protein levels, and consistent with the finding of reduced MYC protein in PRT318-treated and FOXO1-3A-expressing pre-BCR+ cells, all indicating that pre-BCR signaling modulates MYC activity through a mechanism involving SYK and FOXO1. In conclusion, we provide evidence for the dependence of certain B-ALL subgroups on pre-BCR signaling. According to our data this is mainly due to pre-BCR-induced inactivation of FOXO1 and the subsequent deregulation of MYC. Importantly, pharmacological inhibition of pre-BCR signaling with the SYK inhibitor PRT318 completely reversed these effects, therefore providing a rationale for the use of SYK inhibitors in pre-BCR+ subgroups of B-ALL. Figure 1: Figure 1 Figure 1. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership.