Wellik, Linda

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13 C stable isotope resolved metabolomics (SIRM) using U[ 13 C 6 ]Glucose and U[ 13 C 5 ]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13 C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.

Key Points Pembrolizumab was first shown to be clinically active in CLL patients with RT. PD-1 and PD-L1 expression in tumor microenvironment are promising biomarkers to select RT patients for PD-1 blockade.

Key Points Alternatively polarized macrophages are abundant constituents of the tumor microenvironment in T-cell lymphoproliferative disorders. GATA-3 expression identifies a subset of PTCL, NOS with a distinct cytokine profile and inferior survival.

AbstractSTAT3 regulates cell growth by up-regulating downstream targets, such as Myc. The frequency of phosphorylated STAT3 (pSTAT3) and Myc expression and their prognostic relevance is unknown within diffuse large B-cell lymphoma (DLBCL) germinal center B-cell (GCB) and non-GCB subtypes. pSTAT3 and Myc were studied by immunohistochemistry (IHC) on tumors from 40 DLBCL patients uniformly treated on a clinical trial of epratuzumab/rituximab-CHOP. A total of 35% of cases were pSTAT3-positive, and pSTAT3 positivity was more frequent in the non-GCB (P = .06) type but did not correlate with event-free survival (EFS). Myc expression was observed in 50% of cases and was more frequent in non-GCB type (P = .07). Myc-positive cases had inferior EFS in all patients, including the GCB and pSTAT3-positive cases, were more likely to express Myc (P = .06). Myc translocations involving the major breakpoint regions were found in 10% (3 of 29) of cases, and all 3 cases were GCB and had an inferior EFS (P = .09). pSTAT3, but not Myc expression, was correlated with elevated pretreatment serum cytokines, such as IL-10 (P = .05), G-CSF (P = .03), and TNF-α (P = .04). pSTAT3 IHC in DLBCL tumors has the potential to identify patients for STAT3 pathway–directed therapy; Myc IHC is a potential marker for inferior EFS in GCB patients.

Abstract Diffuse Large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma, and is associated with variable clinical presentation as well as variable response to standard chemotherapy regimens. Most significant advances in the treatment of DLBCL over the last 15 years, has been the addition of rituximab to standard chemotherapy. However, still 40% of patients relapse and succumbing to the disease. To improve clinical outcomes for DLBCL patients, it is urgent to identify novel molecular targets and stratify patients according to the expression of specific biomarkers that predict outcomes for the individual patient. Recently, it has been reported that the Signal transducer and activator of transcription 3 (STAT3) is over expressed in DLBCL human cell lines. However, no study has reported the STAT3 expression in DLBCL patient samples or its molecular subtypes (germinal centre B cell type (GCB) and Non-GCB cell type). The phosphorylation of STAT3 (pSTAT3) at its Tyr-705 residue (pSTAT3Tyr705) leads to its activation, followed by dimerization and translocation to the nucleus. Constitutively active STATs (particularly STAT3 and STAT5) contribute to the malignant phenotype in both human cancer cell lines and primary tumors. We hypothesize that constitutive STAT3 activity might be associated with drug resistance and adverse outcome in DLBCL patients. We determined whether STAT3 is constitutively active in patients with DLBCL, and whether activated STAT3 levels correlate with disease severity. Expression of total (tSTAT3) or tyrosine phosphorylated STAT3 (pSTAT3) was determined by in 40 DLBCL tumors. pSTAT3 was found in 52% (21/40) of cases with variable expression {18% low, (+); 25% moderate, (++); and 10% very high (+++)}. However 80% (32/40) of patients expressed tSTAT3. pSTAT3 was expressed in both germinal centre B cells (GCB) (46%) and Non-GCB (69%) DLBCL cases which were molecular subtyped by the Hans method. Furthermore in-vitro inhibition of STAT3 dephosphorylated STAT3 and caused an inhibitory effect on phospho-STAT3 positive DLBCL cells; there was minimal effect on phospho-STAT3 negative cells. Event free survival was lower in pSTAT3 positive compared to pSTAT3 negative DLBCL patients. In summary, our data provide the first evidence that over-expression of pSTAT3 is common in DLBCL, this expression does not clearly relate only to non-GCB tumors and therefore STAT3 inhibitors should be tested in all DLBCL. Pre-treatment assessment of pSTAT3 tumor status will be important to understand whether this IHC marker can preselect patients for response or lack of response to new JAK/STAT targeted agents such as TG101348 (Sanofi Aventis) and BMS-911543 (Bristol-Myers Squibb). Our findings suggest a potential novel therapeutic approach of targeting the inhibition of STAT3 activity in a majority of DLBCL patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2576. doi:1538-7445.AM2012-2576

AbstractCytokines are deregulated in cancers and can contribute to tumor growth. In patients with diffuse large-cell lymphoma (DLBCL), we observed higher levels of JAK/STAT pathway-related serum cytokines (ie, IL-6, IL-10, epidermal growth factor, and IL-2) compared with controls. Of these, only IL-10 activated the JAK2 pathway in lymphoma cells in vitro. Patients with high serum IL-10 had shorter event-free survival (EFS) than patients with low levels (P > .01) and high IL-10 was correlated with high lactase dehydrogenase (P = .0085) and higher International Prognostic Index scores (P = .01). To explore the mechanism by which IL-10 may contribute to an inferior EFS, we investigated the effect of IL-10 on the JAK2 pathway and found that the IL-10/IL-10 receptor complex up-regulated JAK2 signaling. Neutralizing Ab to IL-10 inhibited constitutive and IL-10–induced JAK2/STAT3 phosphorylation. JAK2 inhibition dephosphorylated JAK2 and STAT3 and caused an inhibitory effect on phospho-JAK2–positive DLBCL cells; there was a minimal effect on phospho-JAK2–negative cells. Apoptosis induced by JAK2 inhibition was dependent on inhibition of autocrine IL-10 and c-myc expression and independent of Bcl-2 family expression. These results provide the rationale for testing JAK2 inhibitors in DLBCL patients, and indicate that serum IL-10 may be a biomarker to identify patients more likely to respond to JAK2-targeted therapy.

Abstract The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027–induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027–induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.