ALBERT

Description: The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3. Finally, by introducing mutations designed to disrupt the OCT2–OCA-B interface, we reveal a requirement for this protein–protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell–restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.

Description: Janus kinases (JAKs) classically signal by activating STAT transcription factors but can also regulate gene expression by epigenetically phosphorylating histone H3 on tyrosine 41 (H3Y41-P). In diffuse large B-cell lymphomas (DLBCLs), JAK signaling is a feature of the activated B-cell (ABC) subtype and is triggered by autocrine production of IL-6 and IL-10. Whether this signaling involves STAT activation, epigenetic modification of chromatin, or both mechanisms is unknown. Here we use genetic and pharmacological inhibition to show that JAK1 signaling sustains the survival of ABC DLBCL cells. Whereas STAT3 contributed to the survival of ABC DLBCL cell lines, forced STAT3 activity could not protect these cells from death following JAK1 inhibition, suggesting epigenetic JAK1 action. JAK1 regulated the expression of nearly 3,000 genes in ABC DLBCL cells, and the chromatin surrounding many of these genes was modified by H3Y41-P marks that were diminished by JAK1 inhibition. These JAK1 epigenetic target genes encode important regulators of ABC DLBCL proliferation and survival, including IRF4, MYD88, and MYC. A small molecule JAK1 inhibitor cooperated with the BTK inhibitor ibrutinib in reducing IRF4 levels and acted synergistically to kill ABC DLBCL cells, suggesting that this combination should be evaluated in clinical trials.

Description: Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

Description: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive hematological malignancy associated with the retrovirus human T-cell lymphotropic virus type I. Clinical outcomes of currently available chemotherapies are generally inferior with extremely poor prognosis. Previous studies have recently utilized next generation sequencing technology for the identification of mutated genes that may be pivotal in the pathogenesis of ATLL. However, the identification of indispensable genes for the proliferation and/or survival of ATLL cells remains a formidable challenge due to the complexity of genomic/epigenetic alterations in the ATLL genome. To investigate previously undescribed therapeutic targets in ATLL, we performed a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening to identify genetic vulnerabilities in ATLL cells. Three ATLL cell lines were transduced with lentiviral construct for Cas9 nuclease, followed by lentiviral delivery of the human CRISPR Brunello pooled library (Addgene 73178) of 76,456 single-guide RNAs targeting 19,144 protein-coding genes to cause DNA double-stranded cleavage by the Cas9 nuclease and loss-of-function of the respective genes. Compared with the control cell lines, 23 essential genes, including BATF3 (which we previously discovered by shRNA library screening; Nakagawa et al., Cancer Cell. 34:286-297. 2018) and novel genes (CDK6, JUNB, STAT3, and CCND2) were identified to be involved in ATLL cell proliferation and/or survival. Among these, CDK6 (cyclin-dependent kinase 6), a critical regulatory serine/threonine kinase that forms heterodimers with D-type cyclins, had the best score. The CDK6/D-type cyclin complex regulates E2F transcription factors through the phosphorylation of Rb (retinoblastoma protein), resulting in G1-S transition of the cell cycle. Utilizing publicly available microarray data from peripheral T-cell lymphoma patients, we demonstrated the higher expression of CDK6 in ATLL than that of the other subtypes of T-cell lymphomas, which prompted us to focus on CDK6 as a therapeutic molecular target in ATLL. In confirmatory experiments, two sgRNAs targeting the coding sequences of CDK6 exhibited strong toxicity in five ATLL cell lines in a temporal fashion, which was mediated by G1 cell arrest and partially through apoptosis. We confirmed on-target effect of the sgCDK6 by successfully rescuing cells from toxicity using retroviruses expressing sgRNA-resistant CDK6 cDNA in two ATLL cell lines. The knockout of CDK6 and decrease in the level of phosphorylated Rb were confirmed by immunoblot of sgCDK6-transduced ATLL cell lines. Collectively, the data showed an essential role for CDK6 in cellular proliferation and survival in ATLL. Of the 19,144 genes examined, CDK6 was considered the best vulnerable target for ATLL; therefore, we extended our analysis to evaluate the pharmacological inhibition of CDK6 in ATLL cells. Palbociclib, FDA-approved CDK4/6 inhibitor for breast cancer, was toxic in 11 ATLL cell lines and in four primary ATLL cells but the range of IC50 values were relatively broad (9-6500 nM) among ATLL lines. Because aproximately 20% of ATLL patients carry genetic alteration in a cell cycle/apoptosis regulator TP53 gene, we hypothesized that TP53 alteration may affect the sensitivity of ATLL cells to palbociclib. First, we assessed TP53 status of ATLL cell lines by Sanger sequencing and immunoblotting and showed that six TP53-altered ATLL cell lines exhibited significantly higher IC50 for palbociclib compared with five TP53-intact ATLL cell lines (p

Description: High expression of CC chemokine receptor 4 (CCR4) has been identified as a hallmark gene in ATLL, an aggressive peripheral T-cell neoplasm. CCR4 is a chemokine receptor, which has a critical role in immune cell trafficking including Th2, T-reg and skin-homing memory T-cells. CCR4 ligands, CCL17 and CCL22, were produced in lymph nodes and skin from dendritic cells, macrophages and Langerhans cells. Most ATLL cases express surface CCR4 (90%) and infiltrate to lymph nodes and skin. These observations suggest that CCR4 could have a role in ATLL biology, but it is still unclear whether dysregulation of CCR4 function contributes to ATLL pathogenesis. We performed RNA-Seq for two primary ATLL cases and discovered recurrent non-sense mutations in CCR4. Though an extended analysis using Sanger sequencing, CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. We noticed that the location of the CCR4 mutations in ATLL were reminiscent of mutations affecting the chemokine receptor CXCR4 in the WHIM syndrome, a human immunodeficiency disease. Most CXCR4 mutations in the WHIM syndrome are nonsense or frameshift, resulting in carboxy-terminal truncation of the protein and conferring a gain-of-function phenotype with respect to chemotaxis towards the CXCR4 ligand SDF1. We therefore hypothesized that mutant CCR4 isoforms might enhance chemotaxis of the affected cells to CCR4 ligands. Chemotaxic assay using 32Db, a mouse myeloid cell line, and ED40515(+), an ATLL cell line, clarified that the ectopic expression of CCR4-Q330* enhanced the chemotactic ability of the transduced cells toward CCL17 and CCL22 rather than CCR4-WT transduced cells with statistical significance. To understand the mechanism of this enhanced chemotactic ability, we studied the change in surface CCR4 levels after CCL22 exposure in CCR4-WT-and CCR4-Q330*-reconstituted ED40515(+) cells. Compared with CCR4-WT, CCR4 internalization in CCR4-Q330*-reconstituted cells was significantly impaired. Thus, the ATLL CCR4 mutants impair desensitization by ligand, which likely contributes to the enhanced chemotaxis of cells bearing these mutants. We explored the influence of the ATLL CCR4 mutants on PI(3) kinase (PI3K)-dependent activation of AKT since it has been reported that binding of CCL22 to CCR4 activates AKT in CEM leukemic T-cells and in human Th2 cells. CCR4-Q330*-reconstituted ED40515(+) showed strong activation of AKT with CCL22 ligation compared with CCR4-WT-reconstituted cell. The AKT activation was cancelled with pan-PI3K inhibitor, BKM120, indicating that CCR4-mediated AKT activation was PI3K dependent. Lastly, we tested whether the acquisition of CCR4 mutations by ATLL cells imparts a selective growth advantage relative to cells with wild type CCR4. CCR4-Q330*-reconstituted cells had a selective growth advantage in the presence of CCL22, supporting at least in part the hypothesis that CCR4 mutation are able to provide the affected cells a positive selection pressure through CCL22 ligation and contributes to ATLL pathogenesis. We discovered for the first time recurrent somatic mutations in CCR4 in ATLL. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330 or Y331 in the carboxy-terminus. Functionally, the CCR4-Q330* was a gain-of-function since it increased cell migration towards the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation following receptor engagement by CCL22 in ATLL cells, and conferred a growth advantage in in vitro cultures. Our findings provide a rationale to test whether inhibition of CCR4 signaling might have a therapeutic potential for patients with ATLL. Disclosures No relevant conflicts of interest to declare.

Description: FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase expressed in hematopoietic stem/progenitor cells. Mutated in approximately 1/3 of patients, FLT3 is the most frequently mutated gene in acute myeloid leukemia (AML). The presence of FLT3 internal tandem duplication (ITD) mutations, which renders FLT3 constitutively active, confers a particular poor prognosis. While several FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling, the clinical success of these drugs is limited because they fail to achieve frequent complete durable responses, despite achieving high levels of FLT3 inhibition. These limited clinical responses suggest that monotherapy is unlikely to be curative. Current clinical trials of FLT3 inhibitors are administered in combination with chemotherapy to achieve success in inducing complete remissions. While there is hope that FLT3 inhibition in combination with chemotherapy will increase cure rates, it would be optimal to achieve cures without chemotherapy altogether and its inherent toxicities. Towards this goal it is necessary to uncover critical genes/pathways that collaborate with FLT3 mutations to transform cells. In recent years, the use of whole genome sequencing has greatly expanded the list of genes mutated in AML. However, many of these mutations do not represent a practical therapeutic target because they are present in a low frequency in the patient population, making molecular targeted therapy for each of these improbable to achieve. We hypothesize that these mutated genes likely funnel into a more limited number of signaling pathways, with some pathways being more important than others in contributing to FLT3/ITD AML. Identifying FLT3/ITD cooperative pathways whose inhibition might synergize with FLT3 inhibition could hold promise for greatly improving the cure rate in FLT3 mutant AML. We performed a loss of function RNAi screen in FLT3/ITD+ AML cell lines (Molm14 and MV411) to uncover genes and pathways whose inhibition combined with FLT3/ITD inhibition to more effectively kill FLT3 mutant AML cells. Notch4 signaling was identified as one such potential target. We found Notch4 to be overexpressed in FLT3/ITD+ AML cells lines as well as in the Lin-Sca+Kit+ fraction of bone marrow isolated from FLT3/ITD+ mice. Stable, tetracycline-inducible Notch4 knockdown cell lines were established in Molm14 and MV411 cell lines. While Notch4 knockdown alone slightly decreased cell growth, Notch4 knockdown in combination with FLT3 TKI demonstrated a significant decrease in proliferation compared to FLT3 TKI treated cells expressing a scrambled shRNA control. Additionally, colony forming unit assays revealed that Notch4 knockdown cells treated with FLT3 TKIs exhibited a decrease in clonogenicity compared to FLT3 TKI treated control cells. Pharmacologic inhibition of Notch4 by treatment with gamma secreatse inhibition (GSI) was also tested. Molm14, MV411, and THP-1 (FLT3 wild type AML) cells were treated with FLT3 TKIs (sorafenib, CEP-701, or AC220) alone, GSI alone, FLT3 TKI plus GSI, or vehicle control and assessed for anti-proliferative and apoptotic effects by MTT, and annexin V/7-AAD staining, respectively. In both assays, the combination of FLT3 TKI and GSI exhibited synergy with combination index (CI) values

Description: Abstract 686 Background DLBCL has two molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB), with ABC DLBCL being less curable with current therapy. The survival of ABC but not GCB DLBCL cell lines is sustained by “chronic active” B cell receptor (BCR) signaling. Gain-of-function mutations affecting the BCR subunit CD79B occur in 21% of ABC but only 5% of GCB DLBCL tumors. ABC DLBCL cell lines also depend upon a second pathway for survival that is coordinated by MYD88, an adapter for Toll-like receptors. A constitutively active MYD88 mutant (L265P) is frequent in ABC DLBCL tumors (29%) but rare in GCB DLBCL. CD79B and MYD88 L265P mutations often coexist in ABC DLBCL tumors, suggesting oncogenic collaboration, but can also occur alone. Here, we present interim results of a phase 2 study in relapsed/refractory DLBCL of ibrutinib, a first in class inhibitor of BTK, a kinase in the BCR pathway. We tested the hypothesis that ibrutinib would be more active in ABC than GCB DLBCL due to their different addiction pathways, and we assessed the association of CD79B and MYD88 mutations with response. Methods Subjects with relapsed/refractory de novo DLBCL received ibrutinib 560 mg PO QD. Gene expression profiling (GEP) of formalin-fixed paraffin-embedded biopsy tissues using Affymetrix arrays was used to identify the DLBCL subtype (ABC, GCB, unclassifiable) or were not arrayed (unknown). Sanger sequencing was used to identify CD79B and MYD88 mutations. Subjects underwent CT and PET scanning pre-treatment and every 2 cycles. The primary objective of the study was overall response rate (ORR) categorized by molecular subtype. Response was investigator determined using the revised International Working Group Criteria for NHL. Subjects Seventy subjects were enrolled; median age 63 yrs (28–92); male 71%; stage IV 63%; HI-I/HI IPI 59%; disease ≥10 cm 23%; median prior systemic therapies 3 (1–7); relapsed (27%), refractory (54%) and unknown (19%); prior stem cell transplant 23%; and median time from diagnosis 19 months. Results Safety data are available for 68 subjects who received ≥ 1 dose of ibrutinib. Ibrutinib was well tolerated, with treatment-emergent AEs consistent with data reported in other ibrutinib studies. No new safety signals were identified. Sixty subjects were evaluable per protocol for response (≥ 1 dose of ibrutinib and at least one response assessment). Four subjects in the ABC cohort were not evaluable for response (1 death at study day 12, 1 PD at study day 65, and 2 remain on study treatment but have not had their first response assessment at this analysis). One subject in the GCB cohort was not evaluable for response (death at study day 41). In the ABC subtype, per protocol ORR was 40% (10/25, 95% CI: 21–61%), CR 8% (2/25) and PR 32% (8/25). The median PFS at the time of this analysis is 5.5 months in ABC responders with 60% having not progressed (5 remain on treatment and 1 responder proceeded to transplant). Only one PR was observed in the GCB subtype and none in unclassifiable cases. Thus, ibrutinib showed preferential response activity in ABC versus GCB DLBCL (p=0.0126, Fisher's exact test). Responses occurred in ABC DLBCL tumors with CD79B mutations (60%; 3/5), but also in those with wild type CD79B (37%; 7/19), suggesting that ibrutinib sensitivity does not require a BCR mutation. All cases with both CD79B and MYD88 L265P mutations (n=4) responded, showing that the MYD88 pathway does not prevent ibrutinib activity. In comparison, tumors with only a MYD88 L265P mutation (n=4) did not respond (p=0.0286, Fisher's exact test), suggesting a MYD88-dependent but BCR-independent pathogenesis for some ABC DLBCL cases. Conclusions Ibrutinib showed a clinically meaningful response rate in relapsed/refractory ABC DLBCL, but not in other molecular subtypes. These results are consistent with an essential role of BCR signaling in ABC DLBCL and indicate that future clinical trials of ibrutinib in DLBCL should enroll patients with this subtype. Disclosures: Goy: Pharmacyclics: Research Funding. de Vos:Pharmacyclics: Research Funding. Kenkre:Pharmacyclics: Research Funding. Blum:Pharmacyclics: Research Funding. Advani:Phramacyclics, Inc: Research Funding. Kunkel:Pharmacyclics, Inc: Employment, Equity Ownership. McGreivy:Pharmacyclics, Inc.: Employment, Equity Ownership. Balasubramanian:Pharmacyclics, Inc.: Employment, Equity Ownership. Cheng:Pharmacyclics, Inc.: Employment, Equity Ownership. Moussa:Pharmacyclics, Inc.: Employment, Equity Ownership. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership.