CLL Samples Research Articles

The Influence of the Bone Marrow Niche on Drug Response Phenotypes of Blood Cancers

Background: Signals provided by the microenvironment can modify and circumvent pathway activities that are therapeutically targeted by drugs. However, a systems-level understanding of how the microenvironment and the genetic and molecular alterations of the tumor interact with each other and contribute to drug resistance is lacking.Methods: To address this unmet need, we established an automated microscopy-based phenotyping platform that uses co-culture conditions mimicking the bone marrow environment. We cultured primary tumor cells from more than 100 leukemia patients (CLL, AML, MCL, T-PLL, HCL) with and without bone marrow stroma cell support in DMEM and 10% human serum and treated each condition with 57 drugs in 3 concentrations. After 72h of incubation, 22 000 images per patient were acquired and processed by our custom made image analysis pipeline. Our set-up allows us to increase sensitivity far beyond simple viability testing, as it reads out additional cell type specific features such as cell morphology, autophagy and cell-cell interactions.Results: Quality assessment revealed that in contrast to mono-culture conditions, assay plate edge effects can be avoided under stable stroma cell co-culture conditions. Correlation of replicated patient samples were comparable between mono- and co-cultures (R2>0.75).In the absence of their native microenvironment, primary leukemia cells undergo spontaneous apoptosis ex-vivo. Viability at culture start was always >90% and dropped to a median of 51% (viability range: 17%-90%) after 72h in mono-cultures. Among CLL samples spontaneous apoptosis was not dependent on either IGHV mutation status or any major cytogenetic risk group. Bone marrow stroma cell co-culture conditions protected tumor cells from spontaneous apoptosis (p=8.2e-6, paired t-test). Patient samples with a high degree of spontaneous apoptosis benefited most from co-culture conditions (p=7.2e-10, Pearson correlation).To model interactions of stroma cell conditions and drug-induced apoptosis we established the following linear model: Viability ~ drug-effect + culture-model + drug-effect:culture-model. While activity of some drugs was significantly altered under co-culture conditions, we could also identify drugs with similar activity in mono- and co-cultures. For instance, the activity of common chemotherapeutics (fludarabine: p=0.002 at 0.6µM, cytarabine: p=0.001 at 1.5µM, ANOVA) or bromodomain inhibitors (I-BET-762: p=5.9e-5 at 4.5µM, JQ1: p=1.5e-8 at 1.5µM, ANOVA) was significantly reduced under co-culture conditions. In contrast, PI3K inhibitors idelalisib and duvelisib had a similar activity in mono-culture and stroma co-culture conditions and might represent a starting point to overcome stroma cell mediated drug resistance. In CLL, we identified IGHV mutation status and trisomy 12 as important determinants of response to kinase inhibitors. We confirmed these findings in stroma cell co-cultures, e.g. a better activity of B-cell receptor inhibitors in trisomy 12 and IGHV unmutated CLL. A systematic comparison of ex-vivo drug response pattern in mono- and co-cultures across 171 drug conditions will be presented.Conclusion: Our results suggest that high throughput co-culture drug testing can be robustly performed and provide an unprecedented understanding of how the stroma cell microenvironment and the genetic make-up of tumor cells contribute to drug resistance and sensitivity.Figure: Over 2 million microscopy images were acquired and analysed to assess drug resistance and sensitivity in a co-culture model of primary leukemia and bone marrow stroma cells. blue= Hoechst33342, green=Calcein AM, red=lysosomal dye NIR [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Kinetic Catalytic Inhibition and Cell-Based Analysis, Not Just Target Binding, Are Required to Assess Kinase Selectivity of Covalent Inhibitors: Comparable BTK vs TEC Selectivity Profile for Ibrutinib and Acalabrutinib

▪Background: Currently, kinases are an attractive target class for small molecule inhibitors. However, the high similarity of the ATP binding site among protein kinases presents a significant challenge when developing selective small molecule inhibitors for this target class. Notably, optimal evaluation of covalently bound inhibitors requires consideration of the two-step process involving the initial binding (driven by affinity) and a time-dependent inactivation (driven by covalent bond formation). Accordingly, the kinetic parameters Kinact and Ki are important measures to assess selectivity (Singh et al., Nat Rev Drug Discov, 2011 10:307-317). Furthermore, selectivity evaluated in a cell-based system is useful to confirm kinase occupancy by drug in a more physiologically relevant context. Here we apply three approaches to evaluate the selectivity of two FDA approved drugs, ibrutinib (ibr) and acalabrutinib (aca), against their BTK target and its closely related family member TEC.Methods: Ibr and aca were evaluated in three assays of BTK and TEC activity, with selectivity defined as the ratio of BTK/TEC parameters. For each drug molecule, BTK and TEC biochemical potency was first determined by a mobility shift assay in which substrate phosphorylation was assessed after 3 hr, allowing sufficient time for binding and time-dependent inactivation. IC50 based on drug concentration and percent inhibition was determined accordingly. A second biochemical analysis, also based on a mobility shift assay, was performed in which reaction progression was continuously monitored for 5 hr to determine the kinetic parameters Kinact and Ki. The respective ratios of Kinact/Ki were then used to calculate the BTK/TEC selectivity. A third TEC occupancy assay, similar to a previously described BTK occupancy assay, was further developed to evaluate selectivity between the two kinases in a more physiologically relevant cell lysate system. Both cellular BTK and TEC occupancies were measured after incubation for 0.5, 1, and 3 hr with the two drugs at 8 concentrations, ranging from 0 to 1 µM, using the MWCL-1 cell line and commercially available antibodies. Occupancies of the two kinases by ibr and aca were additionally quantified in 3-month treatment-free human CLL samples after incubation for 3 hr with each drug.Results: The biochemical IC50 values for kinase inhibition demonstrated selectivity of 1.0 and 4.2 fold for ibr and aca, respectively (Table 1). These results indicate much lower selectivity for aca than previously published estimates based on affinity-based kinome profiling alone (Barf, et al, J Pharmacol Exp Ther, 2017, 363:240-252). Aca was also found to be highly potent for TEC with an IC50 of 9.7±2.6 nM. Kinact/Ki determinations for ibr and aca produced nearly equivalent selectivity ratios of 1.5 and 3, respectively, consistent with data published by Liclican et al. (Blood, 2016 128:1594). In the MWCL-1 cell line, similar relative selectivity was also observed in the occupancy assay for ibr and aca, ranging 0.4-1 fold for both compounds across three timepoints. In the four human CLL samples, the relative selectivity for BTK/TEC of ibr was 0.8 and aca was 0.9 (Figure 1).Conclusions: The current data indicate that the selectivity of aca for BTK versus TEC is no more than 3-fold based on kinetic analyses. Cellular target occupancy data further confirm that the BTK and TEC selectivity for ibr and aca is approximately equivalent. Collectively, this study contrasts with prior published reports of BTK/TEC selectivity, demonstrating that selectivity evaluation of these covalent kinase inhibitors based on kinome binding data alone is suboptimal and does not reflect true physiological selectivity when accounting for exposure, binding mechanism, enzymatic activity, and possible protein turn-over in the cellular milieu. [Display omitted] DisclosuresHopper:Pharmacyclics LLC, an AbbVie Company: Employment. Gururaja:Pharmacyclics LLC, an AbbVie Company: Employment, Equity Ownership. Kinoshita:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Dean:CTI BioPharma Corp.: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Equity Ownership. Hill:AbbVie: Equity Ownership; Principia Biopharma: Patents & Royalties: Patent for Principia Biopharma ; Pharmacyclics LLC, an AbbVie Company: Employment. Mongan:Pharmacyclics LLC, an AbbVie Company: Employment, Other: Travel, accommodations, expenses; AbbVie: Equity Ownership; Thermo Fisher Scientific: Patents & Royalties: Patents.

PO-445 E7107 treatment results in aberrantly spliced transcripts and protein products of P53 pathway genes

IntroductionRecent studies indicate that E7107, a spliceosome inhibitor, causes altered splicing of key genes in CLL. We evaluated the effect of E7107 on cell viability and key proteins in the p53 pathway.Material and methodsEight leukaemia/lymphoma cell lines and eight primary CLL samples (6 wild-type and 2 mutant for SF3B1) were exposed to E7107 (H3 Biomedicine) for 72 and 48 hours. Cell viability was assessed by XTT assay. To understand the effect of splicing modulation on key proteins in the p53 pathway, including p21 and MDM2, five B-cell lines were treated with E7107 for 24 hours.Results and discussionsE7107 decreased cell viability at low nanomolar concentrations in all CLL samples (mean LC50=10.5±2.0 nM; but >300 nM in two healthy PBMC controls). No correlation between drug sensitivity and SF3B1 status was observed in CLL samples (p=0.5). Six out of eight cell lines were sensitive to E7107 (mean GI50=6±1.8 nM). The GI50 values were 60.2 and 203.5 nM for the resistant HEL and HAL-01 cells, respectively. The most frequently mutated regions of the SF3B1 gene, exons 14, 15 and 16, had no mutations detected by Sanger sequencing. There was no correlation between drug sensitivity and TP53 status.Western immunoblot revealed a marked decrease of MDM2 protein level in all cell lines, accompanied by a reciprocal concentration-dependent increase in p53. The normal molecular weight p21 disappeared at higher doses of E7107, with concomitant appearance of a high molecular weight p21 isoform (~30 kDa) in TP53 wild-type cells. In addition, a lower molecular weight p53 isoform was seen in OCI-Ly3 cells treated with 50 nM E7107, likely due to alternative splicing. RT-PCR of p21 mRNA, spanning exons 3 to 4, revealed an additional longer transcript in OCI-Ly3 cells treated with E7107(50 nM, 24 hours), which sequencing confirmed was due to intron retention, predicted to result in a high molecular weight protein with an alternative c-terminal sequence. RT-PCR of MDM2 mRNA spanning exons 1 to 11 detected an altered MDM2 transcript with exons 3–10 spliced out.ConclusionE7107 treatment resulted in decreased viability of B-cell lines and primary CLL samples independently of SF3B1 status. This was associated with the production of an aberrant high molecular weight isoform of p21 due to intron retention, and a short isoform of MDM2 missing exons 3–10. Loss of normal MDM2 was accompanied by increased p53. Further investigation is needed to understand the contribution of abnormal isoforms to cell fate.

Abstract 3443: Full-length characterization of transcript isoforms to investigate cancer-associated mutations

Abstract Mutations in the splicing factor SF3B1 in various cancers have been associated with characteristic alterations in splicing. SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 on a junction level have been observed, these patterns have not been systematically examined on an isoform level. Isoform-level analyses are imperative for building a better understanding of altered splicing in a cell; exon connectivity is best resolved using long reads. As such, we have resequenced cDNA from CLL samples with and without K700E mutation in SF3B1, as well as a normal B cell, with nanopore sequencing technology. By converting changes in current caused by blockage of DNA threading through a nanopore into sequence, nanopore sequencing can sequence complete molecules of DNA that exceed 200kb in length. We have developed a novel workflow to perform isoform-level differential splicing analyses leveraging the full-length transcript data that nanopore affords. We report results from nanopore sequencing data that are concordant with known SF3B1 biology from short read sequencing as well as altered splicing patterns that are only observed definitively using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E identifies a bias toward increased alternative 3' splice sites than alternative 5'. In addition, we observe a relative decrease in the expression of genes with intron retention events in CLL samples compared to B cell. We also discover a previously unreported finding of enrichment in intron retention events in SF3B1WT relative to SF3B1K700E and no enrichment between CLL SF3B1MT and B cell, suggesting that mutated SF3B1 in CLL modifies the intron retention landscape to be more similar to a normal phenotype. Gene ontology analyses reveal enrichment in nuclear export in genes associated with intron retention events, and we postulate that many of these are detained introns of transcripts that are kept in the nucleus. With full-length cDNA sequence, we are also able to better detect premature termination codons and estimate the proportion of transcripts subject to nonsense-mediated mRNA decay. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the utility of nanopore sequencing for cancer and splicing research. Citation Format: Alison Tang. Full-length characterization of transcript isoforms to investigate cancer-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3443.

Abstract 2857: PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies

Abstract B-cell lymphoid malignancies, including CLL and DLBCL, are characterized by abnormal activation of Bruton's tyrosine kinase (BTK)-mediated B-cell receptor signaling that contributes to malignant transformation. Ibrutinib (IB) is an oral covalent BTK inhibitor that has shown impressive clinical activity. However, many IB-treated patients relapse over time with fulminant progression. We sought to define the molecular pathways that mediate Ib resistance by studying changes that occurred in vitro in cell lines and primary patient samples with prolonged exposure to IB. We found IB-increased apoptosis in a dose- and time-dependent manner, with upregulated levels of PTEN, BIM, and FOXO3a in Mec-1 and RIVA cells; followed by decreased phosphorylation (p) of Ser473-AKT and Ser253-FOXO3a. mRNA expression levels of Foxo3a and its targets Bim and Pten were significantly upregulated in a dose- and time-dependent manner. Interestingly, a subset of miRNAs, including miRNA-494 were significantly downregulated with upregulation of target transcripts Pten and Bim. Similar results were observed after IB treatment of primary CLL cells of naïve patients and those on new drug targets at high risk for relapse. To examine if miRNA-494-dependent PTEN regulation and activation of PI3K/AKT/FOXO3a signaling has a role in de novo and acquired resistance to IB in B-cell malignancies, we investigated the resistance mechanism that develops to IB therapy by generating IB-resistant (Ib-R) cells. Chronic exposure of RIVA cells to IB demonstrated AKT activation and increased pSer253-FOXO3a, in turn, leading to decreased PTEN and BIM levels; attributed to its decreased transcription as assessed by qRT-PCR in Ib-R RIVA cells. Interestingly, Ib-R RIVA cells had strikingly higher miRNA-494 expression and decreased Pten mRNA levels, indicating further regulation of PTEN/AKT/FOXO3a signaling. Moreover, unlike IB-sensitive CLL samples, patients with partial remission and Ib-R, before and after in vivo IB treatment, demonstrated significant upregulation of miRNA-494 and decreased Pten mRNA expression. The Ib-R cells were sensitized to treatment with IB by PI3K (idelalisib) and AKT (MK-2206) inhibition. MK-2206 downregulated pAKT and sensitized Ib-R cells through AKT-mediated PTEN activation. siRNA-mediated downregulation of AKT and FOXO3a significantly modulated cell survival, demonstrating the importance of these cell survival factors for Ib-R. Importantly, overexpression of miRNA-494 inhibitor increased Pten mRNA levels, further indicating regulation of apoptosis by AKT/FOXO3a signaling. Collectively, these findings reveal a novel mechanism of ibrutinib resistance in B-cell malignancies that modulates the expression and activity of tumor suppressor proteins and pro-apoptotic factors to confer drug resistance that could be effectively inhibited by a rational combination of therapeutic regimens. Citation Format: Yue Li, Isha Kapoor, Huayuan Zhu, Brian T. Hill, Wei Xu, Alex Almasan. PI3K/AKT activation in de novo and acquired resistance to ibrutinib in lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2857.

50. Different cytogenetic culture conditions yield surprising results

Different mitogens added to cytogenetic culture directly influence conventional cytogenetics and FISH results. Especially in the case of cancer patients, the presence or absence of mitogens can yield results with distinct un-related clones, which suggest two different diseases. In our laboratory, CLL samples are cultured for 72 h with one culture stimulated with pokeweed mitogen (PWM), phorbol myristate acetate (PMA), and CpG oligodeoxynucleotide (ODN), and a second culture is stimulated with CpG ODN only. In contrast, B-cell lymphomas are cultured for 72 h with CpG ODN only and cultured for 24 h unstimulated, where myeloid samples are cultured 24 h without any mitogens.

The miR-17/92 cluster is involved in the molecular etiology of the SCLL syndrome driven by the BCR-FGFR1 chimeric kinase.

MicroRNAs have pathogenic roles in the development of a variety of leukemias. Here we identify miRNAs that have important roles in the development of B-lymphomas resulting from the expression of the chimeric BCR-FGFR1 kinase. The miR-17/92 cluster was particularly implicated and forced expression resulted in increased cell proliferation, while inhibiting its function using microRNA sponges reduced cell growth and induced apoptosis. Cells treated with the potent BGJ389 FGFR1 inhibitor led to miR-17/92 downregulation suggesting regulation by FGFR1. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-17/92 expression in stable transduced cell lines demonstrated that BCR-FGFR1 can regulate miR-17/92 expression. This positive association of miR-17/92 with BCR-FGFR1 was also confirmed in primary mouse SCLL tissues and primary human CLL samples. miR-17/92 promotes cell proliferation and survival by targeting CDKN1A and PTEN in B-lymphoma cell lines and primary tumors. An inverse correlation in expression levels was seen between miR-17/92 and both CDKN1A and PTEN in two cohorts of CLL patients. Finally, in vivo engraftment studies demonstrated that manipulation of miR-17/92 was sufficient to affect BCR-FGFR1 driven leukemogenesis. Overall, our results define miR-17/92 as a downstream effector of FGFR1 in BCR-FGFR1 driven B cell lymphoblastic leukemia.

Abstract 1479: Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma

Abstract MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and are frequently aberrantly expressed in cancer. We aimed to understand their role in the transformation of indolent follicular lymphoma (FL) into an aggressive diffuse large B cell lymphoma. This happens in ~3% of cases per year during the course of the disease, and is associated with median survival of only 2 years. The NGS revealed number of aberrations associated with transformed FL (tFL), including frequent high-level activity of MYC (amplifications, translocations, and mutations) or loss of DNA damage regulators (p53, CDKN2A/B). Firstly, we performed a miRNA profiling (TaqMan miRNA Arrays) in paired FL and tFL samples (N=8 pairs). This revealed a relatively small group of 5 miRNAs that are consistently differentially expressed in tFL (P<0.05, fold-change >1.5). Since the most frequently acquired aberration in tFL is the high-level activity of MYC we performed a correlation analysis of MYC levels and expression of these miRNAs in additional samples of FL, tFL, and CLL samples with/without MYC duplication (N=40 FL/tFL, N=39 CLL). This revealed that at least one of these miRNAs is significantly down-modulated (P<0.05) in cases with high-levels of MYC. The MYC-mediated repression of miRNA levels was also observed (P<0.05) in B cells from transgenic MYC over-expressing mice (MYC controlled by an Ig-alpha enhancer) in comparison to wild-type animals (samples obtained from young animals before occurrence of any malignancy). We have further shown that the levels of this miRNA affect B cell proliferation in vitro, and its low-levels associate with percentage of Ki67 positive cells in FL samples (P<0.005). Moreover, low levels of tFL-associated miRNA were present in FL cases with a shorter overall survival (P<0.01), and its expression directly affected BCR signalling (calcium flux assay after anti-IgM). We have shown that the expression of this miRNA is not only down-modulated by high-level MYC expression, but also by B cell adhesion to stromal cells in co-culture in vitro (HS-5 stromal cells). This suggests that its normal physiological function might be related to regulation of B cell functions in the context of immune niches, and this might play a role in FL progression and transformation. It remains to be elucidated what other molecular mechanisms ensure low-level expression of the studied miRNA in cases that do not harbor MYC over-expression, and what pool of target mRNAs is regulated by this miRNA in FL cells.This work was supported by: the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. contact: marek.mraz@email.cz Citation Format: Katerina Musilova, Gabriela Pavlasova, Vaclav Seda, Eva Vojackova, Katerina Cerna, Veronika Svobodova, Robert Pytlik, Vit Prochazka, Zuzana Prouzova, Sarka Pospisilova, Lenka Zlamalikova, Heidi Mocikova, Lenka Kruzova, Marie Jarosova, Andrew Evans, Clive Zent, Leos Kren, Marek Trneny, Jiri Mayer, Andrea Janikova, Marek Mraz. Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2017-1479

Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL.

De novo resistance and rapid recurrence often characterize responses of B-cell malignancies to ibrutinib (IBR), indicating a need to develop drug combinations that block compensatory survival signaling and give deeper, more durable responses. To identify such combinations, we previously performed a combinatorial drug screen and identified the Bcl-2 inhibitor venetoclax (VEN) as a promising partner for combination with IBR in Mantle Cell Lymphoma (MCL). We have opened a multi-institutional clinical trial to test this combination. However, analysis of primary samples from patients with MCL as well as chronic lymphocytic leukemia (CLL) revealed unexpected heterogeneous de novo resistance even to the IBR+VEN combination. In the current study, we demonstrate that resistance to the combination can be generated by microenvironmental agonists: IL-10, CD40L and, most potently, CpG-oligodeoxynucleotides (CpG-ODN), which is a surrogate for unmethylated DNA and a specific agonist for TLR9 signaling. Incubation with these agonists caused robust activation of NF-κB signaling, especially alternative NF-κB, which led to enhanced expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, thus decreasing dependence on Bcl-2. Inhibitors of NF-κB signaling blocked overexpression of these anti-apoptotic proteins and overcame resistance. Inhibitors of Mcl-1, Bcl-xL, or survivin also overcame this resistance, and showed synergistic benefit with the IBR+VEN combination. We conclude that microenvironmental factors, particularly the TLR9 agonist, can generate de novo resistance to the IBR+VEN combination in CLL and MCL cells. This signaling pathway presents targets for overcoming drug resistance induced by extrinsic microenvironmental factors in diverse B-cell malignancies.

Spleen Tyrosine Kinase Is Involved in the CD38 Signal Transduction Pathway in Chronic Lymphocytic Leukemia.

The survival and proliferation of CLL cells depends on microenvironmental contacts in lymphoid organs. CD38 is a cell surface receptor that plays an important role in survival and proliferation signaling in CLL. In this study we demonstrate SYK's direct involvement in the CD38 signaling pathway in primary CLL samples. CD38 stimulation of CLL cells revealed SYK activation. SYK downstream target AKT was subsequently induced and MCL-1 expression was increased. Concomitant inhibition of SYK by the SYK inhibitor R406 resulted in reduced activation of AKT and prevented upregulation of MCL-1. Moreover, short-term CD38 stimulation enhanced BCR-signaling, as indicated by increased ERK phosphorylation. CXCL12-dependent migration was increased after CD38 stimulation. Treating CLL cells with R406 inhibited CD38-mediated migration. In addition, we observed marked downregulation of CD38 expression for CLL cells treated with R406 compared to vehicle control. Finally, we observed a clear correlation between CD38 expression on CLL cells and SYK-inhibitor efficacy. In conclusion, our study provides deeper mechanistic insight into the effect of SYK inhibition in CLL.

Mapping the HLA Ligandome Landscape of Chronic Myeloid Leukemia Identifies Novel CD8+ and CD4+ T Cell-Epitopes for Immunotherapeutic Approaches

While the discovery of BCR-ABL and the respective tyrosine kinase inhibitors (TKI) resulted in a significant prolongation of patient survival rates, there still is no curative treatment for chronic myeloid leukemia (CML) except for allogeneic stem cell transplantation. The concept of T cell-based immunotherapy is a promising opportunity to eliminate residual leukemic cells, which might promote disease relapse after TKI discontinuation. As effective antigen-specific immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens, we have developed a mass spectrometry-based approach, which allows for the direct identification of naturally presented tumor-associated HLA ligands in hematological malignancies. In this study we used this approach to identify HLA class I and II CML-associated peptides as targets for T cell-based immunotherapy.Analysis of HLA class I ligandomes of primary CML cells (n=16) identified 8,291 HLA ligands representing 4,337 source proteins. Comparative ligandome profiling using a benign HLA class I database, which includes various healthy tissues (n=188, 65,949 HLA ligands, 14,030 source proteins) originating from peripheral blood, bone marrow, kidney, lung, liver, colon, spleen and others, revealed 38 CML-exclusive HLA class I ligands with frequencies ≥ 25% of CML patients. Because of the important indirect and direct roles of CD4+ T cells in anti-cancer immune responses, an optimal immunotherapy approach requires the inclusion of HLA class II epitopes. Hence we also analyzed the HLA class II ligandomes of primary CML cells (n=15, 2,822 HLA ligands, 794 source proteins). Comparative ligandome analysis (benign tissue, n=114, 54,149 HLA ligands, 8,584 source proteins) identified 44 CML-associated HLA class II ligands showing CML-exclusive representation in > 25% of the analyzed CML samples.To validate the immunogenicity of our HLA class I and II CML-associated peptides, we performed IFNγ- ELISPOT assays after 12-days of in vitro peptide stimulation. For HLA class II antigens, a panel of 4 peptides was implemented for stimulation of PBMCs obtained from CML patients and healthy volunteers (HV). The ELISPOT assay revealed peptide-specific immune recognition of 4/4 (100%) CML-exclusive peptides in CML patients. The frequencies of the detected immune responses ranged from 17% (4/23 patients) to 4% (1/23 patients) within the tested CML samples. These immune responses were mediated by functional CML patient-derived CD4+ T cells and strictly CML-directed, as no immune response against CML-associated peptides could be detected in HV (0/8).For HLA class I antigens, ELISPOT assays were performed using a panel of 8 peptides. Immune responses were only detected for 1/8 (13%) peptides with a low frequency of 6% (1/18 patients) of tested CML patient samples. A possible explanation for the observed weak immune response to our HLA class I CML-associated peptides compared to the immune responses shown for HLA class II peptides and for HLA class I peptides in other hematological malignancies (e.g. CLL (Kowalewski et. al. PNAS 2015)) might be an inhibition of CD8+ T cell-responses, that reportedly occurs upon TKI treatment of CML patients. To prove this hypothesis in our CML patient cohort (all patients included were under TKI treatment at the time of sample collection), we compared the ELISPOT positive controls (stimulated with a set of 5 Epstein-Barr viral peptides) of all analyzed CML samples with positive controls derived from HV and CLL samples. We could show a highly significant mean spot count reduction (per 100,000 cells) in CML samples (mean 74±16 spots, n=19) compared to HV (mean 241±24 spots, n=42, p<0.001, two-tailed t-test) or CLL (mean 218±16 spots, n=125, p=0.008) samples, confirming the general debilitated CD8+ T cell-response in CML patients under TKI treatment.To prove the immunogenicity of our HLA class I CML-associated peptides, we performed in vitro artificial antigen-presenting cell (aAPC)-based priming experiments of HV CD8+ cells. For one HLA-A*03-restricted peptide we observed tetramer-positive CD8+ populations with frequencies ranging from 0.12% to 1.41% of viable cells in 2/2 HVs so far.Taken together, these results are a first step towards a successful validation of these newly defined HLA class I and II CML-associated antigens as prime targets for further T cell-based immunotherapy approaches in CML patients. DisclosuresKowalewski:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

Identification of Effective Combinations of Targeted Drugs for Hematologic Malignancies

Introduction: Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies represents an ongoing challenge. Large-scale sequencing efforts have identified that many hematologic malignancies, such as acute myeloid leukemia (AML), are driven by a spectrum of mutations and may require combinations of targeted agents to be treated effectively. In addition, the emergence of genetically heterogeneous subclones leading to relapse, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways further necessitate combinatorial therapies. To identify combinations of targeted drugs for AML and other hematologic malignancies, we performed ex vivo profiling of pairs of small-molecule inhibitors for sensitivity against primary patient samples.Methods: Freshly isolated primary mononuclear cells from patients (n=122) with various hematologic malignancies (AML n=58, CLL n=42, ALL n=12, and MPN or MDS/MPN n=10) were cultured in the presence of a panel of 48 drug combinations in equimolar dose series encompassing different classes of compounds, including kinase inhibitors, bromodomain inhibitors, BH3 mimetics, and histone deacetylase inhibitors. For comparison, cells were also tested against graded concentrations of each inhibitor alone, and sensitivity was assessed by MTS-based viability assay. IC50 and AUC values were derived using a probit regression model. Efficacy of each combination relative to its single agents was calculated as a Combination Ratio (CR) value, defined as the combination IC50 or AUC divided by the lowest single agent IC50 or AUC value. A CR value < 1 indicates the combination is more effective relative to the single agent. Associated clinical characteristics were obtained where possible. For the 2 largest diagnostic groups, AML and CLL, expanded panels of clinical, prognostic, mutational, cytogenetic, and surface antigen data were compiled for comparisons according to CR values for each combination.Results: Unsupervised hierarchical clustering of CR values revealed several distinct clusters (Figure 1). Myeloid leukemia patient samples were enriched within a cluster of sensitivity to combinations pairing the Bcl-2 inhibitor venetoclax with select tyrosine kinase inhibitors (dasatinib, doramapimod, sorafenib, or idelalisib). A subset of samples within this cluster showed sensitivity to combinations involving the MEK inhibitor trametinib and a second kinase inhibitor (idelalisib, palbociclib, or quizartinib). In contrast, a discrete subcluster of predominantly lymphoid leukemia patients showed sensitivity to combinations of the histone deacetylase inhibitor panobinostat in tandem with either the JAK inhibitor ruxolitinib or the multi-kinase inhibitor sorafenib. Importantly, apart from venetoclax which as a single agent demonstrated potent and selective efficacy in CLL patient samples, the single agent efficacies do not align selectively to a combination efficacy-derived cluster (Figure 1). Comparison of CR values within each of the 4 diagnostic groups revealed partial overlap in statistically significant effective combinations, while also highlighting unique sensitivities by group, such as idelalisib-quizartinib for AML and ibrutinib-quizartinib for CLL. Further relevant clinical and genetic features were compared within each of the 2 largest groups, AML and CLL. Among AML samples, patients harboring mutations in NPM1 or DNMT3A demonstrated significant sensitivity to combinations of JQ1 and sorafenib (median CR: 0.357) or JQ1 and palbociclib (median CR: 0.119), respectively. AML patients featuring surface expression of CD11b (Integrin aM) or CD58 (LFA-3) were sensitive to combinations of venetoclax and JQ1 or venetoclax and doramapimod, respectively. Among CLL samples, patients harboring deletion of 13q showed significant sensitivity to combinations of palbociclib with either venetoclax or trametinib (median CR: 0.267 and 0.116, respectively).Conclusions: The data reveal multiple specific patterns of ex vivo drug combination efficacy beyond that of either single agent, which are associated with select, actionable diagnostic and genetic subsets, warranting their evaluation in the clinic. These findings highlight the heuristic value of an integrated approach for identifying novel treatment strategies for improved disease control and patient outcomes. [Display omitted] DisclosuresDruker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Tyner:Constellation Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Research Funding; Inctye: Research Funding; Genentech: Research Funding; Aptose Biosciences: Research Funding; Seattle Genetics: Research Funding; Array Biopharma: Research Funding; AstraZeneca: Research Funding; Leap Oncology: Consultancy; Janssen Research & Development: Research Funding.

Targeted Sequencing for Clinical Investigation in Chronic Lymphocytic Leukemia

IntroductionNext generation sequencing (NGS) studies have documented the highly heterogeneous landscape of somatic mutations in CLL. Beside the mere genetic diversity of the disease, frequency and clinical impact of mutations vary between analyzed cohorts since samples may be collected at variable clinical stages. To address this point, we undertook an integrative systematic genomic study of patients included in a prospective clinical trial.Material and methodsWe performed conventional karyotype (K), fluorescence in situ hybridization (FISH) and investigated 31 genes and 2 non-coding regions, in pretreatment samples obtained from 146 patients enrolled on a phase 3 trial comparing fludarabine (F)-cyclophosphamide (C)-rituximab vs. F-C-alemtuzumab (Lepretre et al, Blood 2012). All patients were Binet stage B (n=115) or C (n=31) and devoid of 17p deletion. All gave their informed consent according to the declaration of Helsinki. We studied by targeted deep sequencing exonic regions of 31 genes including TP53, SF3B1, NOTCH1, ATM, BIRC3, FBXW7, NFKBIE, MED12, MYD88 and POT1. DNA was extracted from total blood mononuclear cells (fraction of tumor cells determined by flow cytometry >80%). Targeted regions were PCR-amplified using an AmpliSeq (Life technologies) approach and PCR products sequenced in a MiSeq (Illumina) instrument with a mean depth of 1000X. We used a variant allele frequency (VAF) minimal threshold of 5% to detect mutation. EGR2, PAX5 enhancer and NOTCH1 3’UTR regions were analyzed by Sanger sequencing. For each identified mutation, clinical and biological variables were compared between mutant and wild-type patients using either Wilcoxon or Fisher exact test. For each mutation present in a sufficient number of samples, prognostic impact on overall survival (OS) and progression free survival (PFS) was assessed using log-rank test. For important clinical and molecular prognostic variables, adjusted prognostic effect was assessed using a multivariate Cox model for OS and PFS with a stepwise-forward selection based on Akaike criteria.ResultsChromosomal abnormalities were observed by K in 63% of 126 patients, 11% showing complex K and 23% carrying translocations. FISH revealed 78/144 (54%) 13q deletion, 25/144 (17%) trisomy 12 (tri12), 30/146 (21%) 11q deletion. Unmutated (UM) IGHV status was observed in 84/146 (58%) cases. At least one mutation was observed in 118/146 (81%) patients, with 1, 2, 3 and 4 or 5 mutations in, respectively, 34%, 23%, 17% and 7%. The most frequently mutated genes were SF3B1 (23%), NOTCH1 (16%), ATM (14%), NFKBIE (10%), XPO1 (10%), MED12 (7%), EGR2 (6%), TP53 (5%), BIRC3 (5%), FBXW7 (5%), PAX5 enhancer (5%), CHD2 (5%), PCLO (5%), MYD88 (4%), POT1 (4%), KMTD2 (4%) and NOTCH1 3’UTR (4%). MYD88 and SAMHD1 mutations were always observed as clonal (VAF>40%) whereas BIRC3 and BRAF mutationsalways observed as sub-clonal. NOTCH1 mutations were associated with high CD38 expression (P<.0001), UM IGHV (P<.0001) and tri12 (P=.04); FBXW7 with tri12 (P=.009); NOTCH1 3’UTR with high CD38 (P=.01) and UM IGHV (P=.04). UM IGHV were associated with XPO1 (P=.002) and MED12 mutations (P=.04); mutated IGHV with MYD88 (P=.005) and PAX5 enhancer mutations (P=.01). PAX5 enhancer and POT1 mutations were associated with normal K (P=.02 and .03, respectively). No individual mutant gene carried prognostic significance, but 3 or more mutations were associated with a shorter PFS (HR=2.4 [1.1-5.7]), as UM IGHV (HR=2.2 [1.2-3.9]) and Binet stage C (HR=3.7 [2.1-6.5]). Three or more mutations were associated with a shorter OS (HR=2.3 [1.1-4.7]), as Binet stage C (HR=4.4 [2.1-9.1]), whereas 13q deletion was associated with a longer OS (HR=0.5 [0.2-0.9]). By multivariate analyses, UM IGHV and Binet stage C retained prognostic significance for PFS, and 13q deletion, Binet stage C and 3 or more mutations for OS.ConclusionThese results show that our approach is amendable to routine CLL analyses. They also confirm, in pretreatment CLL samples, the frequency of mutations in NFKBIE, MED12 and EGR2 genes and in PAX5 enhancer and NOTCH1 3'UTR non-coding regions. Some associations were confirmed (eg NOTCH1 and tri12, MYD88 and mutated IGHV). Eighty-one percent of the patients (patients with 17p deletion being excluded) exhibited at least 1 mutation. No individual mutation is associated with a poor prognosis, but the presence of 3 or more mutations is associated with shorter PFS and OS. DisclosuresRibrag:ArgenX: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Infinity: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; NanoString: Membership on an entity's Board of Directors or advisory committees; Pharmamar: Membership on an entity's Board of Directors or advisory committees; Esai: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Knock-Down of the NFAT2 Long and Intermediate Isoforms Leads to CLL Acceleration

NFAT2 is a highly phosphorylated transcription factor which regulates developmental and activation programs in diverse cell types. We and others have previously described a significant overexpression of NFAT2 in CLL cells as compared to physiological B cells. Three major isoforms of NFAT2 with different regulatory properties have been described (700aa short isoform, 800aa intermediate isoform, 900 aa long isoform). Here, we analyzed the role of different NFAT2 transcripts in CLL with respect to disease phenotype and cell proliferation.We investigated primary samples from CLL patients (n=30) for their expression profile of different NFAT2 isoforms using RT-PCR. Applying an shRNA approach, we generated stable knock-down cells of the CLL cell line MEC-1 for the long and intermediate isoforms and for the entire NFAT2 gene resulting in the complete ablation of all isoforms. The proliferation properties of the different MEC-1 cell lines was subsequently assessed in xeno-transplant experiments into NSG mice.While physiological B cells express comparable levels of the short and intermediate/long isoforms, we could detect a five fold overexpression of the intermediate/long isoforms in primary CLL samples. To further analyze the differential regulation of the different NFAT2 transcripts on tumor cell proliferation and cell cycle regulation, we injected NSG mice with MEC-1 cells with intact NFAT2 (n=6), MEC-1 cells with a knock-down of the intermediate and long isoforms (n=6) and MEC-1 cells with a complete NFAT2 knock-down (n=6). MEC-1 cells with selective ablation of the intermediate and long NFAT2 isoforms grew significantly faster in NSG mice than MEC-1 cells with intact NFAT2 expression or MEC-1 cells with a complete NFAT2 knock-down. MEC-1 cells selectively lacking the intermediate and long isoforms led to accelerated tumor proliferation upon subcutaneous injection. Cell cycle analysis as assessed by flow cytometry showed a significantly increased number of cells in the G1/S-Phase for the group without expression of the short isoform, while the group with complete NFAT knock-down exhibited a compromised growth pattern as compared to wild-type MEC-1 cells.In summary, our data demonstrate that genetic loss of the intermediate and long isoforms of NFAT2 leads to CLL acceleration DisclosuresNo relevant conflicts of interest to declare.

IL-4 rescues surface IgM expression in chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia (CLL) cells express poor levels of surface immunoglobulin (sIg), and many are minimally activated or anergic in response to B-cell receptor (BCR) crosslinking in vitro. Paradoxically, CLL cells in patients are highly activated through BCR signaling and expand in proliferation centers, suggesting that the function of sIg signaling is rescued. Here, we find that, compared with normal naïve B cells, CLL cells express a low level of total CD79b protein but normal levels of CD79a and IgM protein. Association of both CD79a and CD79b to IgM is markedly reduced. We further find that interleukin-4 (IL-4) markedly rescues CD79b and sIgM protein in CLL samples. These changes significantly enhance signaling in response to BCR crosslinking. Furthermore, we find that these changes are more pronounced in immunoglobulin heavy chain variable (IGHV)-unmutated CLL cells than IGHV-mutated CLL cells. The results described herein reveal that reduced sIgM is due to low expression of total CD79b protein in CLL cells. IL-4 substantially restores CD79b protein expression, sIgM expression, and BCR signaling.

Abstract 408: ATM function and mutation in CLL 11q deletion samples

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of abnormal B-cell development in which cell death mechanisms have been altered. As the second most common type of leukemia in adults, it progresses more slowly than other types of leukemia and is considered incurable. Approximately 10% of previously untreated patients with CLL exhibit a substantial deletion in the q arm of chromosome 11(22-23), the site of the ATM gene, the product of which regulates homologous recombination repair of double strand DNA breaks (DSB). This lesion occurs at increased incidence, about 50% in patients relapsing on fludarabine-cyclophosphamide-rituximab (FCR) therapy. The residual ATM allele is often mutated, suggesting that the region containing ATM is important for response to chemoimmunotherapy. Although some mutations that cause non-synonymous substitution of amino acids associated with loss of ATM function are known, because the ATM protein contains more than 3,000 amino acids, it is unlikely that all most or all non-synonymous changes would cause loss of function. To address this issue, we developed an immunoblot assay to determine ATM function in del(11q22-23) samples from CLL patients. We confirmed that SMC1 and KAP1 are unique substrates of ATM. Their phosphorylation levels after irradiation (IR) were linearly correlated with ATM activity. This was demonstrated using cell lysates mixtures from cells derived from an individual with ataxia telangiectasia that are deficient in ATM, and those cells repleted with ATM. Consequently, the ratios of phospho to total proteins of SMC1 and KAP1 were employed as indicators of ATM function in response to IR treatment. Using a pool made up of lysates from FISH-negative CLL samples (n = 8) as a positive standard, we validated this assay in 46 del(11q22-23) CLL samples. The phosphorylation ratios of SMC1 and KAP1 from 46 CLL samples were analyzed simultaneously by the normal mixture model-based clustering method, and a formula was generated to determine ATM function. Using this formula, we identified 13 ATM function-deficient samples. To determine whether there is a correlation between ATM gene mutation and the function of the protein, we conducted targeted sequencing of the ATM gene in the 46 samples. Genomic DNA of T-cells isolated and expanded from each of these samples was extracted to serve as a germline reference for the CLL cells. Through a series of bioinformatics analyses, 12 different ATM somatic mutations were identified. Fifteen other ATM mutations were recognized as germ line mutations. No strong correlation was observed between ATM mutation and function. Therefore, alternative assays of ATM function, such as that we have described, are needed to identify ATM deficient tumors in order to develop selective therapies, e.g. agents that cause DSB or inhibit redundant repair pathways. Citation Format: Yingjun Jiang, Xiaojun Liu, Hsiang-Chun Chen, Kim-Anh Do, Xiaoping Su, William Wierda, Michael Keating, William Plunkett. ATM function and mutation in CLL 11q deletion samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 408.

Chromatin accessibility maps of chronic lymphocytic leukaemia identify subtype-specific epigenome signatures and transcription regulatory networks.

Chronic lymphocytic leukaemia (CLL) is characterized by substantial clinical heterogeneity, despite relatively few genetic alterations. To provide a basis for studying epigenome deregulation in CLL, here we present genome-wide chromatin accessibility maps for 88 CLL samples from 55 patients measured by the ATAC-seq assay. We also performed ChIPmentation and RNA-seq profiling for ten representative samples. Based on the resulting data set, we devised and applied a bioinformatic method that links chromatin profiles to clinical annotations. Our analysis identified sample-specific variation on top of a shared core of CLL regulatory regions. IGHV mutation status—which distinguishes the two major subtypes of CLL—was accurately predicted by the chromatin profiles and gene regulatory networks inferred for IGHV-mutated versus IGHV-unmutated samples identified characteristic differences between these two disease subtypes. In summary, we discovered widespread heterogeneity in the chromatin landscape of CLL, established a community resource for studying epigenome deregulation in leukaemia and demonstrated the feasibility of large-scale chromatin accessibility mapping in cancer cohorts and clinical research.

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR.

TP53 Mutations and Other Cell-Intrinsic Determinants of the Apoptotic Response to Ibrutinib in Chronic Lymphocytic Leukemia

Abstract Introduction: Ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p (all lines). Overall response rates to ibrutinib in treatment naïve or relapsed CLL are high and most patients experience prolonged remission durations, although cases of acquired ibrutinib resistance have been reported. Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment cell interactions. Weakening of these interactions underlies some of the mechanisms by which ibrutinib is working in vivo. However, comparatively little is known about CLL cell-intrinsic determinants of apoptotic responses induced by ibrutinib. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance and ultimately clinical outcome are warranted. Methods: We first characterized 50 paired CLL samples procured before and after standard CLL therapies (predominantly chemo immunotherapies) for IGVH status, ZAP70 positivity, CD38 expression, TP53 mutations and FISH and analyzed the relapsed samples by whole exome sequencing (WES). Preliminary ibrutinib treatment studies using CD40-expressing fibroblasts or IL-4 or both in CLL co-culture resulted in low ibrutinib activity, precluding measurements of cell-intrinsic response determinants. Therefore, to assay ibrutinib-mediated CLL cell kill, CLL samples were purified using negative selection and incubated in RPMI medium supplemented with 10% FCS for 72 hours with ibrutinib at varying doses (0.25 µM - 5 µM). Cell death and viability was measured using AnnexinV-PI staining and flow cytometry. IC50 values were calculated using the program XLfit. Results: CLL cells demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.37 µM - 9.69 µM in pre-treatment samples and 0.56 µM - &gt;10 µM in post treatment samples (mean IC50 values for pre-treatment and relapsed samples were 2.7 µM and 3.7 µM, respectively). Multiple cell-intrinsic determinants of IC50 values to ibrutinib were identified; CLL samples from IGVH unmutated samples were more sensitive than samples from IGVH mutated samples (mean IC50 values for pre-treatment samples were (IGVH -UM) 2.1 µM and (IGVH -M) 3.5 µM (p=0.01), and relapsed samples of (IGVH -UM) 2.8 µM and (IGVH -M) 4.0 µM (p=0.03), respectively, consistent with a greater dependency/signaling capacity of the BCR in IGVH-UM CLL. Similar findings were derived for ZAP70 status, with CLL samples with &lt;20% ZAP70 positivity by FACS being less sensitivity to ibrutinib than ZAP70 positive samples (mean IC50 values for relapsed CLL samples were 4.1 µM and 2.8 µM, respectively; p=0.03). Trisomy 12 was also associated with sensitivity to ibrutinib (mean IC50 values for trisomy 12 positive rCLL samples were 2.8 µM and 4.1 µM, respectively; p=0.03). IC50 results for paired samples demonstrated a strong concordance within paired samples (Pearson correlation coefficient 0.66). However, ~10% of CLL samples were substantially more resistant to ibrutinib at relapse when compared with their paired diagnosis samples. Of these 5 samples, three had acquired mutations in TP53 following prior chemotherapy; a hypothesis-generating finding that acquired TP53 mutations reduce ibrutinib sensitivities. An expansion cohort of 7 additional ex vivo ibrutinib-treated TP53-mutated CLL samples confirmed the novel and clinically relevant finding that TP53 mutated CLL cells are substantially less sensitive to ibrutinib than TP53 wild type cells (mean IC50 values of 6.1 µM and 2.7 µM, respectively; p&lt;0.001) Conclusion: Ibrutinib ex-vivo treatment of 50 paired CLL samples procured before and after standard CLL therapies identified cell-intrinsic determinants of ibrutinib sensitivity, including IGVH unmutated CLL and trisomy 12 and implicates prior chemotherapy-selected mutations in TP53 as an intrinsic cause of reduced response. Expansion data identified TP53 mutations as a cell-intrinsic ibrutinib resistance factor in CLL. This novel finding may explain observed shorter remission durations for CLL patients with del17p/mTP53 undergoing ibrutinib therapy. Ex vivo data overall appear concordant with findings in patients treated with ibrutinib thus externally validating our approach and may allow for further refinements in ibrutinib therapy and response prediction. Disclosures Balasubramanian: Pharmacyclics LLC, an AbbVie Company: Equity Ownership; Janssen: Employment, Equity Ownership. Malek:Janssen Pharmaceuticals: Research Funding; Abbvie: Equity Ownership; Gilead Sciences: Equity Ownership.

Biological Significance of B Cell Receptor Mediated Regulation of Autophagy in Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of B cells which is broadly split into two groups representing a progressive IGHV unmutated (U-CLL) and a more indolent IGHV mutated (M-CLL) disease. Activation of the B cell receptor (BCR) by antigen/autoantigen engagement is crucial for CLL cell survival, disease progression and resistance to therapy, however further research is required to better understand how BCR signalling impacts on CLL biology. Autophagy is known to play a role in tumorigenesis and resistance to therapy in solid tumors, however whether autophagy has a role in CLL biology and how it is regulated has not been fully investigated. Autophagy is important for normal B cell development and is known to be regulated by various drug treatments in vitro in CLL samples. A previous study showed that activation of the BCR on murine splenic B cells with soluble or bead immobilised (BI) anti-IgM induced autophagy and subsequent apoptosis, however, the role of BCR-induced autophagy has not been explored in B cell malignancies and particularly CLL. Firstly, we assessed basal protein expression of key autophagy markers LC3BII, and ATG3 in CLL samples and age-matched normal donor B cells (NDB). CLL cells expressed significantly more LC3BII (p=.014, n=57) and ATG3 (p=.04, n=58) compared with NDB (n=8), with a greater LC3BII protein expression in U-CLL compared to M-CLL (p=.039, n=57), indicating more autophagy occurs in U-CLL. Furthermore basal increases in autophagy markers GABARAPL2 (LC3B family member) (p=.0004, n=34) and ATG4A (p=.04, n=20) at the RNA level were significantly associated with the ability of CLL cells to flux calcium (&gt;10%) in response to anti-IgM. This indicated a possible role of the BCR in the regulation of autophagy in CLL samples and a possible association with progressive disease. Activation of the CLL BCR with BI anti-IgM significantly induced expression of autophagy markers ATG3 (p=.002, n=22), LC3BII (p&lt;.0001, n=23) and p62 (p=.0011, n=9) at the protein level and ATG3 (p=.04, n=8) and GABARAPL2 (p=.03, n=6) at the RNA level in a time dependent manner. Next, CLL samples were treated with BI anti-IgM in the presence or absence of the autophagy inhibitor hydroxychloroquine (HCQ) to confirm the increase in autophagic flux indicated at the RNA level. Addition of HCQ in combination with BI anti-IgM significantly increased accumulation of LC3BII protein expression compared with HCQ or anti-IgM alone (p=.01 and p=.02 respectively, n=5) indicating an increase in autophagic flux. Treatment with ibrutinib or R406 prevented BI anti-IgM dependent increases in LC3BII (p=.02 and p=.009 respectively, n=9) and p62 expression (p=.02 and p=.006 respectively, n=9). Confirming the role of the BCR in the regulation of autophagy in CLL samples and indicating that inhibition of autophagy may contribute to the clinical responses seen with these BCR kinase inhibitors. Finally we showed that BI anti-IgM increased CLL cell viability compared to BI isotype control (p=.002, n=7), whilst the addition of HCQ significantly reversed this effect (p=.001, n=7). More importantly treatment with BI anti-IgM protected CLL cells against fludarabine induced cell death (p=.0004, n=3) which again was reversed by the addition of HCQ (p=.05, n=3). These data emphasise the importance of BCR signalling in the regulation of autophagy and its impact on therapy resistance and suggest a possible role for autophagy inhibitors in the treatment of CLL. Disclosures Strefford: Roche: Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.