ALBERT

Description: Many studies have shown that deletion at chromosome 17p targeting the TP53 gene, or del(17p), is associated with poor prognosis in chronic lymphocytic leukemia (CLL). Despite this, not all del(17p) CLL progresses rapidly to treatment and has short survival. We hypothesized that other coexisting genetic aberrations may contribute to the poor clinical outcome and heterogeneity of del(17p) cases. To assess this, we analyzed copy number alterations (CNAs) using Affymetrix SNP array data from 200 CLL patients (55 with del(17p)), and somatic mutation profile by whole exome sequencing (WES; Illumina) in 168 patients (53 with del(17p)). Ninety-nine patients were studied with both SNP arrays and WES, 39 of whom had del(17p). Analysis of copy number showed that del(17p) CLL had a median of 11 CNA events, mostly copy number losses, compared to 2 events in WT CLL (p=1.1E-12). Both the number of CNA events (p=7.9E-10) and the total length of copy number gain (p=1.6E-4) or loss (p=1.3E-8) were associated with shorter overall survival (OS), even when controlling for co-existing del(17p). Using GISTIC analysis, we discovered three significantly deleted regions specific to del(17p), namely large chromosomal deletions at 3p, 4p, and 9p. These novel recurrent deletions were rarely seen in wild type CLL and the presence of any of these deletions with del(17p) was strongly associated with shorter OS compared to del(17p) alone. We were able to evaluate complex karyotype (CKT) determined by stimulated metaphase cytogenetics in a subset of the cohort, and found that 17/27 (63%) evaluable del(17p) CLLs had CKT, while only 13/58 (22%) WT CLLs did (p=0.0005). Analysis of OS showed that del(17p) and CKT together had worse OS than either individually, although individually they each conferred OS worse than WT (p

Description: In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008). Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p

Description: Constitutive activation of the NF-κB pathway is a critical feature of hematologic malignancies and is associated with increased lymphocyte proliferation and survival. Various mechanisms leading to altered NF-κB activation have been described in lymphomas, including activating mutations in and upregulation of NF-κB pathway genes. We have previously reported whole-exome sequencing results from a cohort of 160 CLL patients showing mutations in several NF-κB pathway genes (as defined in the Ingenuity Pathway Analysis database), including MYD88 (n=12), RIPK1 (n=4), NRAS (n=4), KRAS (n=3), CARD11 (n=1), IRAK4 (n=1), PIK3CA (n=1) and TRAF3 (n=1). Here, we describe functional approaches to evaluate the biological significance of NF-κB pathway mutations. Using the CellTiter-Glo assay in serum-free conditions, we assessed the relationship between NF-κB pathway mutation status and survival of CLL cells in vitro. We found that CLL cells with no NF-κB mutations exhibited spontaneous apoptosis in vitro, with 3.3 - 39.5% viable cells remaining after 48 hours in culture (n=4 patient samples; n=5 replicates per patient). Similarly, cells harboring the MYD88 L265P mutation yielded 0.6 - 23.1% viable cells after 48 hours (n=5 patient samples; n=5 replicates per patient). In contrast, cells harboring RIPK1 Q375*, RIPK1 K559R, KRAS Q61H, CARD11 E756K, IRAK4 K400E, and PIK3CA I143V displayed apoptotic resistance, with 48.6 - 132.5% viable cells remaining after 48 hours (1 patient sample per mutation; n=5 replicates per patient). Similar findings were observed in the context of B-cell receptor activation via IgM stimulation, with IgM stimulation generally enhancing CLL cell viability (mean = 24% increase after 48 hours). In the case of RIPK1 Q375*, however, no further increase in viability was observed, suggesting that this truncating mutation may obviate the need for external survival signals. To identify NF-κB mutations that might associate with susceptibility to different therapies, we examined the relationship between NF-κB pathway mutation status and sensitivity to the NF-κB inhibitor, SN50. Treatment with SN50 for 48 hours resulted in loss of viability in cells that were negative for NF-κB pathway mutations (43.3 - 98.9% decrease in viability with 5 μM SN50; n=4 patient samples; n=5 replicates per patient). Similarly, cells harboring the MYD88 L265P mutation responded to SN50 treatment (43.1 - 97.0% decrease in viability; n=5 patient samples; n=5 replicates per patient). In contrast, cells harboring RIPK1 Q375*, RIPK1 K559R, CARD11 E756K, and PIK3CA I143V were more resistant to SN50 treatment (18.6% increase - 38.9% decrease in viability; 1 patient sample per mutation; n=5 replicates per patient). These results suggest that specific NF-κB pathway mutations confer resistance to NF-κB inhibition. The above results prompted the question of whether NF-κB pathway mutations may also confer resistance to the BTK inhibitor, ibrutinib, which has previously been shown to block NF-κB pathway activation by inhibiting the phosphorylation of p65 and preventing its nuclear translocation. To address this question, we treated cells with 2.5 μM ibrutinib for 48 hours. Ibrutinib treatment led to a loss in viability in cells that had no NF-κB pathway mutations (26.3 - 82.9% decrease in viability; n=4 patient samples; n=5 replicates per patient). Cells harboring MYD88 L265P mutations also appeared to be susceptible to ibrutinib (25.8 - 82.9% decrease in viability; n=5 patient samples; n=5 replicates per patient). Notably, cells harboring the RIPK1 Q375* and KRAS Q61H mutations appeared to be more resistant to treatment (7.0% decrease and 2.0% increase in viability, respectively; 1 patient sample per mutation; n=5 replicates per patient). Furthermore, RIPK1 Q375* and KRAS Q61H cells remained resistant to ibrutinib in the context of BCR activation. Though our study utilized a limited number of patient samples representing a variety of mutations, the results are suggestive that specific NF-κB pathway mutations are functional and may influence intrinsic CLL cell survival, responsiveness to IgM stimulation, and sensitivity to drug treatment. Identification of specific mutations that confer resistance to ibrutinib is of particular clinical interest for predicting response and understanding drug resistance. Disclosures: Brown: Novartis: Consultancy; Vertex: Consultancy; Sanofi Aventis: Consultancy; Onyx: Consultancy; Emergent: Consultancy; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Pharmacyclics: Consultancy; Genzyme: Research Funding.

Description: Several recent next generation sequencing studies have uncovered mutations in key genes that contribute to CLL pathogenesis. Most CLLs with deletions of chromosome 13q (del(13q), incidence 50-60%) or normal cytogenetics (incidence 15-20%) do not carry these recurrent driver mutations. Clues to the genetic basis of disease in this more indolent group likely lie elsewhere in the genome. We present preliminary data from whole-genome sequencing (WGS) of a cohort of CLL patients with del(13q) (n=13) and normal cytogenetics (n=10), sequenced at 30X and 60X coverage for tumor and normal (saliva) respectively. Of the 23 patients, 43% were male and the median age at diagnosis was 53 years. The median time from diagnosis to sampling was 1.9 years, and 22 of 23 patients were previously untreated. With a median follow-up of 4.8 years in the overall cohort, 8 patients (35%) had been treated at a median 2.5 years from diagnosis. Although indolent by cytogenetics and of low Rai stage (median 1), the cohort includes balanced numbers of ZAP70± and IGHV mutated/unmutated cases. A total of 68,928 point mutations with an average of 2,995 mutations per genome were detected in our cohort using the MuTect algorithm, which is approximately 60% fewer mutations than in multiple myeloma. The pattern of mutations in protein coding regions was similar to previous exome sequencing studies, with an average of 18.7 ±7.4 non-synonymous mutations per case (0.57±0.2 per Mb). The rate of mutations was significantly lower in patients diagnosed at a younger age (p=0.016, 2 sided t test). As expected based on the cytogenetic makeup of our cohort, the incidence of mutations in the 20 known CLL driver genes did not reach genome wide significance. However, the incidence of mutations in these driver genes was significantly higher in patients with normal cytogenetics as compared to del(13q) (60% vs 18%, p=0.039). Rearrangement events were identified using the dRanger algorithm followed by mapping of the precise breakpoint to single base pair resolution using the Breakpointer algorithm. A total of 78 rearrangements were identified, corresponding to a median of 3 (range 0-15) per CLL genome, significantly fewer than in most other cancers. Similar to other hematologic malignancies, the incidence of deletions (70%) was significantly higher than that of tandem duplications (9%) (Wilcoxon signed rank test, p=0.0001). In addition, inter-chromosomal translocations exhibited a strong positive correlation with long range deletions (Pearson’s r=0.72, p=0.0001) and a moderate positive correlation with inversions (Pearson’s r=0.64, p=0.0009), suggesting a common mechanism. The only clinical feature associated with the presence of structural rearrangements was ZAP70 positivity (p=0.046). Of the 13 patients with del(13q), 4 were due to inter-chromosomal rearrangement that led to loss of the common minimal region of deletion. The other most recurrent rearrangement partners were chr.2 (n=12 cases, 52%), chr.14 (n=9 cases, 39%) and chr.1 (n=5 cases, 21%), while no rearrangements were seen in chromosomes 8,11,17,18 and 19. The remaining chromosomes were affected in

Description: The L265P somatic mutation in the Myeloid Differentiation Primary Response 88 (MYD88) gene is recurrently observed in CLL; although this mutation has been demonstrated to have functional effects in multiple hematologic malignancies, its role in CLL is largely unknown. To address this gap in knowledge, we examined the clinical and biological impact of MYD88 L265P mutations in CLL by analysis of gene expression, cell viability and Toll-like Receptor 9 (TLR9)-induced signaling and cytokine production. Out of 160 CLL patient samples subjected to whole-exome sequencing and previously reported by our group, 10 were found to harbor MYD88 L265P mutations, all of which possessed mutated immunoglobulin heavy chain variable (IGHV) regions (p = 0.006). While IGHV mutated patients are generally known to exhibit better prognosis compared to IGHV unmutated patients, the presence of MYD88 L265P within the IGHV mutated subset was associated with earlier age of disease onset (p = 0.04) and worse overall survival (OS; p = 0.00017), comparable to IGHV unmutated samples with wild-type (WT) MYD88. No association with the presence of chromosome 13q deletions (p = 0.26) or prior treatment at the time of sampling (p = 0.10) was observed. Gene expression microarray analysis restricted to the IGHV mutated subset (MYD88 WT: n = 76; MYD88 L265P: n=10) and conducted using a PAM-based approach demonstrated that MYD88 L265P mutation was associated with differential expression of 28 genes, whose expression was then examined across all CLL samples with available gene expression data (n = 150). Using Cox modeling, a composite gene signature score was determined for each patient, who were subsequently dichotomized based on median signature. This method was able to predict both OS and event free survival (EFS) in a univariable analysis (OS: p = 1.2E-06; EFS: p = 7.6E-13). Statistical significance was maintained when a multivariable analysis was conducted, adjusting for known CLL risk factors including age, IGHV status, ZAP70 expression, cytogenetics and prior treatment (p 〈 0.0001 for OS and EFS). The univariable (OS: p = 1.6E-05; EFS: p = 5.7E-10) and multivariable findings (p 〈 0.003 for OS and EFS) were further confirmed in an independent validation cohort (n = 87). To identify a more parsimonious gene set, we applied a L1 penalized proportional hazards model to the discovery and validation cohorts, separately. This approach identified 5 overlapping genes (BCAT1, BMP6, CHAD, IKZF2, and TRIO) between the two cohorts that appear to be the main drivers of the predictive signature. To inhibit MYD88 signaling in CLL cells, we treated MYD88 L265P and WT cells (n = 6/group, matched for clinical characteristics: IGHV, ZAP70, cytogenetic, and treatment status) in vitro with a highly-selective small molecule IRAK4 inhibitor, ND-2158 (Nimbus Therapeutics). ND-2158 significantly reduced cell viability in a dose dependent manner in both MYD88 WT and L265P primary CLL cells, either alone or in combination with a fixed concentration of the B-cell receptor (BCR) pathway inhibitor, ibrutinib. The TLR9 agonist CpG was used to stimulate signaling through the MYD88 pathway in vitro. ND-2158 inhibition of CpG-induced IRAK4 activation in CLL cells (n = 3/group, matched for clinical characteristics) blocked IRAK1 and IκBα degradation and led to a dose-dependent decrease in the ratio of phospho/total proteins. No significant differences were noted between MYD88 WT and L265P samples, consistent with our cell viability results. CLL-secreted levels of IL-6, IL-10 and CCL3 were measured in culture supernatants treated with ND-2158+/- CpG stimulation (n = 4/group, matched for clinical characteristics). CpG stimulated cytokine levels (p 〈 0.0001 for all cytokines+/- CpG) were significantly inhibited in a dose-dependent manner by ND-2158. Again, no significant differences were observed between MYD88 WT and L265P CLL with respect to cytokine production, either at baseline or in CpG-stimulated DMSO treated control cells. In conclusion, the differences in clinical outcome and gene expression observed between MYD88 WT and L265P IGHV mutated CLLs indicate a functional role for MYD88 L265P in CLL. The inferior clinical outcome in IGHV mutated CLL with L265P mutation suggests that MYD88 signaling may be a relevant target in CLL. ND-2158 inhibits signaling in the MYD88 pathway, suggesting potential therapeutic utility of IRAK4 inhibitors in CLL. Disclosures Chaudhary: Nimbus Therapeutics: Equity Ownership. Miao:Nimbus Therapeutics: Employment. Westlin:Nimbus Therapeutics: Employment.

Description: Recent work in CLL and other lymphoproliferative disorders has established the importance of somatic mutations in the pathogenesis and prognosis of disease. MYD88 L265P mutations occur in approximately 30% of ABC diffuse large B cell lymphomas (ABC DLBCL) and 90% of Waldenstrom’s macroglobulinemia (WM) cases and have been shown to be an activating mutation in both cancers. MYD88 is an important adaptor protein for IL-1 and many Toll-like receptors, which signal via dimerization followed by formation of a complex of MYD88 with IRAK4 and IRAK1, leading to activation of IRAK4 kinase activity, IRAK1 phosphorylation and downstream activation of NFκB. Previous work in CLL has demonstrated that MYD88 L265P mutations occur in a smaller subset of patients (2.9-6.25%) and although MYD88 mutation is thought to be a CLL driver mutation, its role in CLL is not clearly understood. We previously reported whole exome sequencing results from a cohort of 160 CLL patients, 10 of whom have MYD88 L265P mutation (6.25%). Eight of ten patients with MYD88 L265P mutation had mutated IGHV, consistent with prior reports of this association. However, patients with MYD88 L265P mutation did not have a younger age at diagnosis, possibly due to the overall younger median age of our patient cohort (54, range 34-77). RNA gene expression profiling of 146 CLL and 20 normal B cell samples revealed that normalized median expression of IRAK1, IRAK4 and NFKB1 appear lower in CLL samples versus normal B cells (IRAK1 p = 0.0001, IRAK4 p = 0.01, NFKB1 p = 0.0005). However, median expression levels of MYD88 appear higher in CLL samples versus normal B cells (p = 0.006). Although MYD88 L265P mutation has been shown to activate BTK in WM, mean BTK protein levels measured by flow cytometry were similar between CLL patients with and without MYD88 mutation (mean % cells positive for BTK, 98.3% in MYD88 L265P mutants vs. 98.16% in wild-type [WT], 3 patients/group), as were levels of phosphorylated BTK (mean % cells positive, 13.28% in MYD88 L265P vs. 14.35% in WT). Chronic BTK-mediated B cell receptor signaling is a hallmark of CLL and studies with the BTK inhibitor ibrutinib (PCI-32765) have demonstrated encouraging therapeutic results in vitro and in vivo. ND-2110 and ND-2158 are potent, highly selective IRAK4 kinase inhibitors which have single agent activity in vitro in ABC DLBCL cell lines as well as synergistic effects with BTK inhibitors. Given these findings, ND-2110 and ND-2158 were ideal candidates to test in CLL. As such, CLL samples with and without MYD88 L265P mutation (3 per group) were cultured in vitro in the presence of 5μM ND-2110 or ND-2158 as single agents or in combination with 2.5μM ibrutinib for 72 hours. A second, higher dose combination was also tested, using 25μM ND-2110 or ND-2158 and 3.75μM ibrutinib. Viability was assessed using Cell TiterGlo and normalized to DMSO treated cells. A general linear model was used to assess the association of % viability with MYD88 L265P mutation, IgM stimulation and combinations of IRAK4 inhibitor compounds and ibrutinib. MYD88 L265P was associated with higher viability, hence less cell killing (p = 0.002 5μM ND-2110 & 2.5μM ibrutinib; p 〈 0.0001 5μM ND-2158 & 2.5μM ibrutinib; p = 0.005 25μM ND-2158 & 3.75μM ibrutinib). Cells treated with 25μM ND-2158 + 3.75μM ibrutinib had significantly lower viability compared to cells treated with 25μM ND-2158 alone (p 〈 0.0001). IgM stimulation also resulted in lower viability compared to no stimulation, suggesting sensitization (p = 0.002 25μM ND-2100 + 3.75μM ibrutinib; p 〈 0.0001 all other combinations). Ibrutinib appeared to have less single agent activity in MYD88 L265P mutants, while the activity of single agent ND-2110 and ND-2158 was more equalized between groups. These preliminary results are currently being confirmed in additional patients. To further characterize the effects of these drugs, we have established a model system in which CLL cells are co-cultured with NIH3T3 cells expressing CD40L, with and without CpG stimulation. Preliminary results show that in one WT MYD88 CLL patient, these conditions resulted in significant induction of Ki67 expression, which was reduced by combination of either ND-2110 or 2158 with ibrutinib. These results suggest that IRAK4 inhibitors in combination with ibrutinib have interesting activity in CLL, which may differ based on MYD88 L265P mutation; experiments are in progress in additional patients to further characterize this effect. Disclosures: Chaudhary: NIMBUS Discovery: Employment. Romero:NIMBUS Discovery: Consultancy, Equity Ownership. Robinson:NIMBUS Discovery: Consultancy. Westlin:NIMBUS Discovery: Employment. Brown:Pharmacyclics: Consultancy; Genentech: Consultancy; Celgene: Consultancy, Research Funding; Emergent: Consultancy; Onyx: Consultancy; Sanofi Aventis: Consultancy; Vertex: Consultancy; Novartis: Consultancy; Genzyme: Research Funding.