ALBERT

Description: TheKLHL14gene acquires frequent inactivating mutations in mature B cell malignancies, especially in the MYD88L265P, CD79B mutant (MCD) genetic subtype of diffuse large B cell lymphoma (DLBCL), which relies on B cell receptor (BCR) signaling for survival. However, the pathogenic role of KLHL14 in DLBCL and its molecular function are largely unknown. Here, we report that KLHL14 is in close proximity to the BCR in the endoplasmic reticulum of MCD cell line models and promotes the turnover of immature glycoforms of BCR subunits, reducing total cellular BCR levels. Loss of KLHL14 confers relative resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and promotes assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, which initiates prosurvival NF-κB activation. Consequently, KLHL14 inactivation allows MCD cells to maintain NF-κB signaling in the presence of ibrutinib. These findings reinforce the central role of My-T-BCR–dependent NF-κB signaling in MCD DLBCL and suggest that the genetic status of KLHL14 should be considered in clinical trials testing inhibitors of BTK and BCR signaling mediators in DLBCL.

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: The activated B-cell–like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on chronic active B-cell receptor (BCR) signaling. BCR pathway inhibitors induce remissions in a subset of ABC DLBCL patients. BCR microclusters on the surface of ABC cells resemble those generated following antigen engagement of normal B cells. We speculated that binding of lymphoma BCRs to self-antigens initiates and maintains chronic active BCR signaling in ABC DLBCL. To assess whether antigenic engagement of the BCR is required for the ongoing survival of ABC cells, we developed isogenic ABC cells that differed solely with respect to the IgH V region of their BCRs. In competitive assays with wild-type cells, substitution of a heterologous V region impaired the survival of three ABC lines. The viability of one VH4-34+ ABC line and the ability of its BCR to bind to its own cell surface depended on V region residues that mediate the intrinsic autoreactivity of VH4-34 to self-glycoproteins. The BCR of another ABC line reacted with self-antigens in apoptotic debris, and the survival of a third ABC line was sustained by reactivity of its BCR to an idiotypic epitope in its own V region. Hence, a diverse set of self-antigens is responsible for maintaining the malignant survival of ABC DLBCL cells. IgH V regions used by the BCRs of ABC DLBCL biopsy samples varied in their ability to sustain survival of these ABC lines, suggesting a screening procedure to identify patients who might benefit from BCR pathway inhibition.

Description: Abstract 3156 T cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers associated with mutations in NOTCH1. The Growth factor independent-1 (Gfi1) transcriptional repressor gene was originally discovered as a common target of Moloney murine leukemia virus (MMLV) proviral insertion in murine T-ALL. In fact, the Gfi1 locus is the most frequently activated gene in MMLV-induced T cell leukemia. Therefore, we investigated whether the most commonly activated gene in MMLV-induced murine T-ALL, Gfi1, could collaborate with the most commonly activated gene in human T-ALL, NOTCH1. Here, we show that GFI1 expression is associated with Notch signaling in human T-ALL (p'0.0003). Functionally, Gfi1 collaborates with Notch-induced murine T-ALL by accelerating an already rapid disease model (p=0.03) without altering the lymphoblastic nature of the disease. Furthermore, inducible deletion of Gfi1 is counter-selected in both Notch-driven retroviral and transgenic mouse models of T-ALL; whereas, constitutive absence of Gfi1 completely prevents transgenic Notch-induced T-ALL (p≤0.04). However, T-ALL tumors can form in Gfi1-/- animals using either ENU-mutagenesis or MMLV-infection, yet tumor formation is delayed (p≤0.02, p≤0.03 respectively). This suggests that Gfi1 deletion does not prevent the formation of the T-ALL initiating cell and that Gfi1 might be absolutely required for Notch-induced T-ALL. Most striking is that Gfi1 is required for T-ALL maintenance in vitro and in vivo. Using three separate Tal1-initiated murine T-ALL cell lines, the overexpression of the Gfi1 dominant-negative, Gfi1N382S, was quickly and completely counter-selected. As Gfi1 has previously been found to regulate pro-apoptotic genes in T cells, we attempted to rescue the above loss of function phenotype by overexpressing the anti-apoptotic factor Bcl2. Notably, counter-selection of Gfi1N382 is not observed or is significantly delayed in all three cell lines. In vivo, inducible deletion of Gfi1 leads to both mutagen- or Notch-induced tumor regression as measured by ultrasound. In fact, levels of Gfi1 expression directly correlate to tumor regression and disease free survival of T-ALL. Finally, targeting Gfi1 enhances the efficacy of radiation therapy and bone marrow transplantation. Deletion of Gfi1 sensitizes T-ALL tumors and T cells to p53-dependent apoptosis after exposure to DNA-damaging agents such as radiation, Etoposide or Daunorubicin by de-repression of the pro-apoptotic Gfi1 target gene Bax. These data extend the role of Gfi1 to human T-ALL and suggest that T-ALL is dependent upon Gfi1 to repress pro-apoptotic genes for tumor survival, ultimately highlighting a new therapeutic target in the fight against lymphoid malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points ATF3 inhibits lipopolysaccharide-driven CXCL1 production by airway epithelia. ATF3 controls neutrophil recruitment to the wild-type lung and chemotaxis in vitro via TIAM2 expression.

Description: Genomic analyses of diffuse large B cell lymphoma (DLBCL) are revealing the genetic and phenotypic heterogeneity of these aggressive lymphomas. In part, this heterogeneity reflects the existence of distinct genetic subtypes that acquire characteristic constellations of somatic genetic alterations to converge on the DLBCL phenotype. In parallel, functional genomic screens and proteomic analyses have identified multiprotein assemblies that coordinate oncogenic survival signaling in DLBCL. In this review, we merge these recent insights into a unified conceptual framework with implications for the design of precision medicine trials in DLBCL.

Description: Cancer genome and transcriptome sequencing can identify novel oncogenes and tumor suppressors, discover distinct cancer subtypes, and predict therapeutic responses. Analysis of the coding genome of diffuse large B cell lymphoma (DLBCL) has identified various genetic mutations in the ABC and GCB molecular subtypes. Notably, ABC DLBCLs have recurrent activating mutations involving the canonical NF-kB pathway. Although an oncogenic role for constitutive NF-kB activity has been demonstrated in ABC DLBCL, the molecular mechanisms of various oncogenic mutations are still elusive. To understand the pathogenesis of DLBCL, we used high-throughput exome and transcriptome sequencing of more than 500 DLBCL biopsies. To enable functional screening of mutant alleles identified by exome-seq and RNA-seq, we are constructing an inducible retroviral expression library of over 200 open reading frames (ORFs) of genes mutated in DLBCL. We have cloned the mutant and wildtype forms of these genes together with a unique 26-base-pair 'bar code' to facilitate screening with high-throughput sequencing. We have transduced a subset of this ORF library into DLBCL cell lines, induced ORF expression by doxycycline, and FACS sorted into high and low populations based on expression the known NF-κB target gene CD83. We then performed barcode sequencing to assay relative enrichment of ORFs that confer higher or lower NF-κB activity. ORF screening revealed that MYD88L265P, CARD11L232LI, both well-characterized gain-of-function mutants, were among the highest ranking genes for induction of NF-κB activity. Interestingly, we observed enrichment for USP7D271E in NF-κB high populations, though these genes have not been shown to have a tumorigenic role in DLBCL. Ubiquitin Specific Protease-7 (USP7) is a regulator of NF-κB transcriptional activity, in part by de-ubiquitinating p65. To test how USP7 mutations contribute to NF-κB activity, we engineered the ABC DLBCL cell line TMD8 to express an NF-κB reporter consisting of the NF-κB transcriptional response element (TRE) fused to GFP. NF-κB activity upon overexpression of wildtype or mutant USP7 was monitored by flow cytometry (FACS). We observed that the mutated form of USP7 significantly increased NF-κB activity over that seen with wildtype, suggesting this is a gain-of-function mutant. We next used the CRISPR gene-targeting system to probe the function of USP7 genetically. Single guide RNAs (sgRNAs) targeting the USP7 coding regions were coexpressed in TMD8 cells with the endonuclease Cas9. SgRNA-expressing, GFP+ cells were monitored over time among total live cells by flow cytometry, and a relative reduction of the GFP+ population was observed in the sgUSP7 population. These results suggest that USP7 may function as an oncogene in DLBCL. This ongoing work demonstrates the efficacy of a high-throughput ORF expression screen to characterize mutations found in the genomic landscape of the DLBCL. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2174 Growth factor independent-1 (Gfi1) is a zinc finger transcriptional repressor protein originally identified in a rodent model of T-cell leukemia. Gfi1 deficient mice have defects in T cell development and a moderate loss of thymic cellularity. In Drosophila, orthologs of Notch1 and Gfi1 cooperate to specify embryo sensory organ precursors. Given the established requirement for Notch1 in T cell specification and development as well as the functional relationship of Notch and Gfi1 orthologs in Drosophila genetics, we investigated the ability of Gfi1 and Notch to cooperate in T-cell development. Utilizing transgenic mice in which the expression of Cre recombinase is controlled by the proximal Lck promoter (LckCre) to both activate intracellular Notch1 (ICN) while simultaneously deleting Gfi1, we demonstrate that T cells overexpressing ICN require Gfi1 for their survival and proper integration of ICN signaling. First, we validated our approach by showing that Lck-Cre-mediated deletion of Gfi1 alleles (Gfi1flox/-) or activation of ICN expression (Rosa26lox-stop-loxICN ires eGFP, “RosaICN”) lead to expected phenotypes. We next examined the consequences of ICN activation with simultaneous deletion of Gfi1. Whereas inducible deletion of Gfi1 alone decreases thymic cellularity by ∼4-fold, Gfi1 deletion coupled with ICN activation leads to complete thymic involution with a 14-fold reduction in total T cell numbers (p

Description: Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer of aberrant B-lymphocytes. Although a portion of DLBCL is curable with standard immunochemotherapy, patients who fail this treatment have a poor prognosis. Recently, cancer genomics has paved the way for better understanding of the genetic basis of lymphoma pathogenesis. Characterization of point mutations and structural alterations has uncovered novel molecular targets for lymphoma therapy and provided a comprehensive view of lymphoma development. By performing multiplatform genomic analysis of DLBCL biopsy samples, we have identified KLHL14 as a recurrent target of somatic mutations in activated B-cell-like (ABC) DLBCL biopsies (10.8% of patients). KLHL14 contains a BTB (broad complex, tramtrack, and bric a brac) domain that can potentially mediate dimerization and binding to Cullin3 (CUL3)-a essential scaffold component of the Cullin-RING-based E3 ubiquitin ligase complexes. KLHL14 also contains kelch repeats that can form a B-propeller tertiary structure that can serve as a substrate-binding domain. KLHL14 is highly expressed in B-cells but is found at low levels in non-immune tissues. Deficiency of KLHL14 in mice leads to embryonic lethality while KLHL14 heterozygous mice show reduction of B-1a cells, suggesting a role for KLHL14 in B-cell homeostasis. Importantly, KLHL14 mutations are highly enriched in tumors belonging to the recently defined MCD (MYD88L265P/CD79B mutation) genetic subtype of DLBCL, the subset of ABC DLBCLs. Somatic mutations primarily localize to the N-terminus of the protein in the BTB domain and BACK (BTB and C-terminal Kelch) domain. However, the impact of these mutations as well as the molecular function of KLHL14 is largely unknown. To investigate the biological effect of KLHL14 loss of function, we used an inducible CRISPR/Cas9 system to delete KLHL14 in ABC DLBCL cell lines and monitored cell growth. Ablation of KLHL14 resulted in an increase in cell proliferation and survival, supporting a role for KLHL14 as a tumor suppressor. Next, we performed a multiplatform -omic analysis (proteomics, phosphoproteomics, ubiquitinomics, high-throughput sequencing) to explore the signaling networks and interactome of KLHL14. Whereas ectopic expression of wild-type KLHL14 altered the dynamics of tyrosine phosphorylation and ubiquitylation events in ABC DLBCL lines, KLHL14 lymphoma-associated mutant alleles had little if any effect, suggesting that they are loss-of-function variants. Gene expression profiling by RNA-sequencing revealed that KLHL14-inactivated cells have a higher NF-kB target gene expression than wild-type cells. Thus, tumor-associated inactivating mutations of KLHL14 depend on a subset of essential NF-kB-related oncoproteins for their survival and this might contribute to the proliferative advantage of DLBCL. In summary, we have uncovered a tumor suppressive function of KLHL14 and found that KLHL14 mutants promote ABC DLBCL survival by increasing NF-kB activity. These findings suggest that tumors with KLHL14 inactivating mutations may serve as a marker of resistance to anti-NF-kB treatment and provide the basis for treating MCD subtype patients with downstream NF-kB pathway inhibitors in the clinical settings. Disclosures Staudt: Nanostring: Patents & Royalties.