The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy1. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling2,3, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt’s lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, theMYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

Background Pancreatic ductal adenocarcinoma utilizes the CCL2/CCR2 chemokine axis to facilitate recruitment of tumor associated macrophages to sculpt an immunosuppressive tumor microenvironment. This pathway has prognostic implications in pancreas cancer, and blockade of CCR2 restores anti-tumor immunity in pre-clinical models. This provided the rationale for a clinical study in pancreatic adenocarcinoma to determine the safety and recommended phase 2 oral dosage of the CCR2 inhibitor PF-04136309 in combination with chemotherapy (FOLFIRINOX). Methods In this single-center, open label, phase Ib clinical trial patients age ≥ 18 years with treatment naïve borderline resectable or locally advanced, biopsy-proven pancreatic ductal adenocarcinoma, Eastern Cooperative Oncology Group performance status <2, measurable disease by Response Evaluation Criteria in Solid Tumors Version 1.1, and normal end organ function were eligible for enrollment. FOLFIRINOX (oxaliplatin, 85 mg/m2; irinotecan, 180 mg/m2; leucovorin, 400 mg/m2, and bolus fluorouracil 400 mg/m2 followed by 2,400 mg/m2 46 hour continuous infusion) was administered every 2 weeks for a total of six treatment cycles. To determine the recommended phase 2 dose, PF-04136309 was orally administered at a starting dose of 500 mg twice daily in a standard 3+3 dose de-escalation design with an expansion phase planned at the recommended phase 2 dose. Both FOLFIRINOX and PF-04136309 were simultaneously initiated with a total treatment duration of 3 months. The primary endpoints were to determine the recommended phase 2 dose and toxicity of PF-04136309 in combination with FOLFIRINOX. All patients in the dose de-escalation and expansion phase received the recommended phase 2 dose of PF-04136309 were combined for assessment of treatment toxicity by an intention to treat analysis. For tissue specimen comparison in corollary studies, a group of patients receiving FOLFIRINOX alone were enrolled and evaluated for treatment related toxicity. This study has been completed and is registered at ClinicalTrials.gov; number NCT01413022. Results From April 19th, 2012 through November 12th, 2014 a total of 47 patients were enrolled. The dose de-escalation group (n=6) received PF-04136309 at 500 mg administered orally twice daily. No dose-limiting toxicities were observed and this was established as the recommended phase 2 dose. The expansion phase cohort (n=33) and patients in the dose de-escalation arm receiving PF-04136309 at the recommended phase 2 dose (n=6) were combined for assessment of treatment related toxicity. No therapy related deaths occurring during the study interval. Early termination as the result of treatment related toxicity occurred in 2 of the 39 patients (5%) in the FOLFIRINOX plus PF-04136309 arm. Grade ≥3 adverse events reported in ≥10% of the patients receiving PF-04136309 included neutropenia in 27 patients (69%), febrile neutropenia in 7 patients (18%), lymphopenia in 4 patients (10%), diarrhea in 6 patients (15%), and hypokalemia in 7 patients (18%). Among...

RalGEFs were recently shown to be critical for Ras-mediated transformed and tumorigenic growth of human cells. We now show that the oncogenic activity of these proteins is propagated by activation of one RalGEF substrate, RalA, but blunted by another closely related substrate, RalB, and that the oncogenic signaling requires binding of the RalBP1 and exocyst subunit effector proteins. Knockdown of RalA expression impeded, if not abolished, the ability of human cancer cells to form tumors. RalA was also commonly activated in a panel of cell lines from pancreatic cancers, a disease characterized by activation of Ras. Activation of RalA signaling thus appears to be a critical step in Ras-induced transformation and tumorigenesis of human cells.

Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission1. Equal distribution of mitochondria to daughter cells during mitosis requires fission2. Mitotic mitochondrial fission depends upon both the relocalization of large GTPase Drp1 to the outer mitochondrial membrane and phosphorylation of S616 on Drp1 by the mitotic kinase cyclin B/Cdk12. We now report that these processes are mediated by the small Ras-like GTPase RalA and its effector RalBP1 (RLIP76/RLIP1/RIP1)3,4. Specifically, the mitotic kinase Aurora A phosphorylates S194 of RalA, relocalizing it to the mitochondria, where it concentrates RalBP1 and Drp1. Furthermore, RalBP1 associates with cyclin B/Cdk1 kinase activity to foster phosphorylation of Drp1 on S616. Disrupting either RalA or RalBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B/Cdk1 converge upon RalA and RalBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.

Ras is mutated to remain in the active oncogenic state in many cancers. As Ras has proven difficult to target therapeutically, we searched for secreted, druggable proteins induced by Ras that are required for tumorigenesis. We found that Ras induces the secretion of cytokine IL6 in different cell types, and that knockdown of IL6, genetic ablation of the IL6 gene, or treatment with a neutralizing IL6 antibody retard Ras-driven tumorigenesis. IL6 appears to act in a paracrine fashion to promote angiogenesis and tumor growth. Inhibiting IL6 may therefore have therapeutic utility for treatment of cancers characterized by oncogenic Ras mutations.Supplemental material is available at http://www.genesdev.org.

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RalA function is critical to tumor initiation, whereas RalB function is more important for tumor metastasis in the tested cell lines and thus argues for critical, but distinct, roles of Ral proteins during the dynamic progression of Ras-driven pancreatic cancers.

Histone deacetylase inhibitors (HDACI) are promising antitumor agents. Although transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanism and targets by which HDACIs achieve their antitumor effects remain poorly understood. It is not known whether any of the HDAC members support robust tumor growth. In this report, we show that HDAC6, a cytoplasmic-localized and cytoskeletonassociated deacetylase, is required for efficient oncogenic transformation and tumor formation. We found that HDAC6 expression is induced upon oncogenic Ras transformation. Fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several cancer cell lines reduces anchorage-independent growth and the ability to form tumors in mice. The loss of anchorage-independent growth is associated with increased anoikis and defects in AKT and extracellular signal-regulated kinase activation upon loss of adhesion. Lastly, HDAC6-null mice are more resistant to chemical carcinogen-induced skin tumors. Our results provide the first experimental evidence that a specific HDAC member is required for efficient oncogenic transformation and indicate that HDAC6 is an important component underlying the antitumor effects of HDACIs. [Cancer Res 2008;68(18):7561-9]