Aberrant activation of AXL may drive progression of squamous cell carcinoma in CLL patients: a mechanistic study with clinical implications.

BACKGROUND: Occurrence of squamous cell carcinoma (SCC) even in early-stage, untreated chronic lymphocytic leukemia (CLL) patients can be a significant morbidity issue with occasional transformation into metastatic skin lesions. METHODS: CLL cells and extracellular vesicles (EVs) from CLL patients' blood/plasma were purified and used. Expression/activation of AXL and its functions in normal keratinocytes (HEKa) were assessed in vitro co-culture system and in SCC tissues. RESULTS: We detected aberrant activation of AXL, AKT and ERK-1/2 in SCC cell lines compared to HEKa. We also detected increased expression of AXL in primary SCC tissues obtained from CLL patients. Increased activation of AXL, AKT, ERK-1/2 and Src was discernible in HEKa upon co-culturing with CLL cells. Further analysis suggests that Gas6, a ligand of AXL, regulates AXL activation in co-cultured HEKa. Interestingly, exposure of HEKa cells to CLL plasma-derived EVs induced expression of AXL, P-AKT, and EMT-associated markers leading to migration of the cells. Finally, pharmacologic inhibition of AXL induced cell death in SCC lines in a dose dependent manner. CONCLUSIONS: Our findings that CLL cells likely are involved in driving SCC progression, at least in part, via activation of the AXL signaling axis, indicating that AXL inhibition may be beneficial for our CLL patients with SCC.

Dependence of peripheral T-cell lymphoma on constitutively activated JAK3: Implication for JAK3 inhibition as a therapeutic approach.

Peripheral T-cell lymphoma (PTCL) is a clinically heterogeneous group that represents 10%-15% of all lymphomas. Despite improved genetic and molecular understanding, treatment outcomes for PTCL have not shown significant improvement. Although Janus kinase-2 (JAK2) plays an important role in myeloproliferative neoplasms, the critical role of JAK isoforms in mediating prosurvival signaling in PTCL cells is not well defined. Immunohistochemical analysis of PTCL tumors (n = 96) revealed high levels of constitutively active JAK3 (pJAK3) that significantly (p < 0.04) correlated with the activation state of its canonical substrate STAT3. Furthermore, constitutive activation of JAK3 and STAT3 positively correlated, at least in part, with an oncogenic tyrosine phosphatase PTPN11. Pharmacological inhibition of JAK3 but not JAK1/JAK2 significantly (p < 0.001) decreased PTCL proliferation, survival and STAT3 activation. A sharp contrast was observed in the pJAK3 positivity between ALK+ (85.7%) versus ALK-negative (10.0%) in human PTCL tumors and PTCL cell lines. Moreover, JAK3 and ALK reciprocally interacted in PTCL cells, forming a complex to possibly regulate STAT3 signaling. Finally, combined inhibition of JAK3 (by WHI-P154) and ALK (by crizotinib or alectinib) significantly (p < 0.01) decreased the survival of PTCL cells as compared to either agent alone by inhibiting STAT3 downstream signaling. Collectively, our findings establish that JAK3 is a therapeutic target for a subset of PTCL, and provide rationale for the clinical evaluation of JAK3 inhibitors combined with ALK-targeted therapy in PTCL.

Inflammatory Cells in Nephrectomy Tissue from Patients without and with a History of Urinary Stone Disease.

BACKGROUND AND OBJECTIVES: Urinary stone disease has been associated with inflammation, but the specific cell interactions that mediate events remain poorly defined. This study compared calcification and inflammatory cell patterns in kidney tissue from radical nephrectomy specimens of patients without and with a history of urinary stone disease. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Nontumor parenchyma of biobanked radical nephrectomy specimens from age- and sex-matched stone formers (n=44) and nonstone formers (n=82) were compared. Calcification was detected by Yasue staining and inflammatory cell populations by immunohistochemistry for CD68 (proinflammatory M1 macrophages), CD163 and CD206 (anti-inflammatory M2 macrophages), CD3 (T lymphocytes), and tryptase (mast cells). Calcifications and inflammatory cells were quantified in cortex and medulla using Image-Pro analysis software. RESULTS: Calcification in the medulla of stone formers was higher than in nonstone formers (P<0.001). M1 macrophages in the cortex and medulla of stone formers were greater than in nonstone formers (P<0.001), and greater in stone former medulla than stone former cortex (P=0.02). There were no differences in age, sex, body mass index, tumor characteristics (size, stage, or thrombus), vascular disease status, or eGFR between the groups. M2 macrophages, T lymphocytes, and mast cells did not differ by stone former status. There was a correlation between M1 macrophages and calcification in the medulla of stone formers (rho=0.48; P=0.001) and between M2 macrophages and calcification in the medulla of nonstone formers (rho=0.35; P=0.001). T lymphocytes were correlated with calcification in the cortex of both nonstone formers (rho=0.27; P=0.01) and stone formers (rho=0.42; P=0.004), whereas mast cells and calcification were correlated only in the cortex of stone formers (rho=0.35; P=0.02). CONCLUSIONS: Higher medullary calcification stimulated accumulation of proinflammatory rather than anti-inflammatory macrophages in stone formers.

Distinct immune signatures in chronic lymphocytic leukemia and Richter syndrome.

Richter syndrome (RS) refers to transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma. RS is known to be associated with a number of genetic alterations such as TP53 and NOTCH1 mutations. However, it is unclear what immune microenvironment changes are associated with RS. In this study, we analyzed expression of immune checkpoint molecules and infiltration of immune cells in nodal samples, and peripheral blood T-cell diversity in 33 CLL and 37 RS patients. Compared to CLL, RS nodal tissue had higher PD-L1 expression in histiocytes and dendritic cells (median 16.6% vs. 2.8%, P < 0.01) and PD1 expression in neoplastic B cells (median 26.0% vs. 6.2%, P < 0.01), and higher infiltration of FOXP3-positive T cells (median 1.7% vs. 0.4%, P < 0.01) and CD163-positive macrophages (median 23.4% vs. 9.1%, P < 0.01). In addition, peripheral blood T-cell receptor clonality was significantly lower in RS vs. CLL patients (median [25th-75th], 0.107 [0.070-0.209] vs. 0.233 [0.111-0.406], P = 0.046), suggesting that T cells in RS patients were significantly more diverse than in CLL patients. Collectively these data suggest that CLL and RS have distinct immune signatures. Better understanding of the immune microenvironment is essential to improve immunotherapy efficacy in CLL and RS.

Increased glutathione utilization augments tumor cell proliferation in Waldenstrom Macroglobulinemia.

Metabolic reprogramming is a hallmark of cancer cells. In Waldenstrom Macroglobulinemia (WM), the infiltration of IgM-secreting lymphoplamacytic cells into the bone marrow (BM) could shift the homeostasis of proteins and metabolites towards a permissive niche for tumor growth. Here, we investigated whether alerted metabolic pathways contribute to the pathobiology of WM and whether the cytokine composition of the BM promotes such changes. Metabolomics analysis on WM patients and normal donors' serum samples revealed a total of 75 metabolites that were significantly altered between two groups. While these metabolites belonged to amino acids, glucose, glutathione and lipid metabolism pathways, the highest number of the differentially expressed metabolites belonged to glutathione metabolism. Proteomics analysis and immunohistochemical staining both confirmed the increased protein levels mediating glutathione metabolism, including GCLC, MT1X, QPCT and GPX3. Moreover, treatment with IL-6 and IL-21, cytokines that induce WM cell proliferation and IgM secretion, increased gene expression of the amino acid transporters mediating glutathione metabolism, including ASCT2, SLC7A11 and 4F2HC, indicating that cytokines in the WM BM could modulate glutathione metabolism. Glutathione synthesis inhibition using Buthionine sulphoximine (BSO) significantly reduced WM cells proliferation in vitro, accompanied with decreased NFκB-p65 and MAPK-p38 phosphorylation. Moreover, BSO treatment significantly reduced the tumor growth rate in a WM xenograft model, further highlighting the role of glutathione metabolism in promoting tumor growth and proliferation. In summary, our data highlight a central role for glutathione metabolism in WM pathobiology and indicate that intervening with the metabolic processes could be a potential therapy for WM patients.

Targeting glycogen synthase kinase 3 for therapeutic benefit in lymphoma.

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and ß are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3ß compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3ß genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3ß binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3ß is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3ß correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Prognostic and therapeutic significance of phosphorylated STAT3 and protein tyrosine phosphatase-6 in peripheral-T cell lymphoma.

Peripheral T cell lymphomas (PTCL) is a heterogenous group of non-Hodgkin lymphoma and many patients remain refractory to the frontline therapy. Identifying new prognostic markers and treatment is an unmet need in PTCL. We analyzed phospho-STAT3 (pSTAT3) expression in a cohort of 169 PTCL tumors and show overall 38% positivity with varied distribution among PTCL subtypes with 27% (16/59) in PTCL-NOS; 29% (11/38) in AITL, 57% (13/28) in ALK-negative ALCL, and 93% in ALK-pos ALCL (14/15), respectively. Correlative analysis indicated an adverse correlation between pSTAT3 and overall survival (OS). PTPN6, a tyrosine phosphatase and potential negative regulator of STAT3 activity, was suppressed in 62% of PTCL-NOS, 42% of AITL, 60% ALK-neg ALCL, and 86% of ALK-pos ALCL. Loss of PTPN6 combined with pSTAT3 positivity predicted an infwere considered significantferior OS in PTCL cases. In vitro treatment of TCL lines with azacytidine (aza), a DNA methyltransferase inhibitor (DNMTi), restored PTPN6 expression and decreased pSTAT3. Combining DNMTi with JAK3 inhibitor resulted in synergistic antitumor activity in SUDHL1 cell line. Overall, our results suggest that PTPN6 and activated STAT3 can be developed as prognostic markers, and the combination of DNMTi and JAK3 inhibitors as a novel treatment for patients with PTCL subtypes.

Aurora kinase and FGFR3 inhibition results in significant apoptosis in molecular subgroups of multiple myeloma.

Aberrant expression of proteins involved in cell division is a constant feature in multiple myeloma (MM), especially in high-risk disease. Increasingly, therapy of myeloma is moving towards individualization based on underlying genetic abnormalities. Aurora kinases are important mediators of cell cycle and are up regulated in MM. Functional loss of Aurora kinases results in genetic instability and dysregulated division leading to cellular aneuploidy and growth arrest. We investigated the role of Aurora kinase inhibition in MM, using a small molecule inhibitor A1014907. Low nanomolar A1014907 concentrations induced aneuploidy in MM cell lines independent of underlying cytogenetic abnormalities by inhibiting Aurora Kinases. However, A1014907 induced more pronounced and dose dependent apoptosis in cell lines with t(4;14) translocation. Translocation t(4;14) is observed in about 15% of patients with MM leading to constitutive activation of FGFR3 in two-thirds of these patients. Further investigation of the mechanism of action of A1014907 revealed potent FGFR3 pathway inhibition only in the sensitive cell lines. Thus, our results show that aurora kinase inhibition causes cell cycle arrest and aneuploidy with minimal apoptosis whereas inhibiting both aurora kinase and FGFR3 activity induced potent apoptosis in MM cells. These results support clinical evaluation of A1014907 in MM patients with t(4;14) translocation and/or FGFR3 expression.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies.

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 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]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 13C 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.

Pembrolizumab in patients with CLL and Richter transformation or with relapsed CLL.

Chronic lymphocytic leukemia (CLL) patients progressed early on ibrutinib often develop Richter transformation (RT) with a short survival of about 4 months. Preclinical studies suggest that programmed death 1 (PD-1) pathway is critical to inhibit immune surveillance in CLL. This phase 2 study was designed to test the efficacy and safety of pembrolizumab, a humanized PD-1-blocking antibody, at a dose of 200 mg every 3 weeks in relapsed and transformed CLL. Twenty-five patients including 16 relapsed CLL and 9 RT (all proven diffuse large cell lymphoma) patients were enrolled, and 60% received prior ibrutinib. Objective responses were observed in 4 out of 9 RT patients (44%) and in 0 out of 16 CLL patients (0%). All responses were observed in RT patients who had progression after prior therapy with ibrutinib. After a median follow-up time of 11 months, the median overall survival in the RT cohort was 10.7 months, but was not reached in RT patients who progressed after prior ibrutinib. Treatment-related grade 3 or above adverse events were reported in 15 (60%) patients and were manageable. Analyses of pretreatment tumor specimens from available patients revealed increased expression of PD-ligand 1 (PD-L1) and a trend of increased expression in PD-1 in the tumor microenvironment in patients who had confirmed responses. Overall, pembrolizumab exhibited selective efficacy in CLL patients with RT. The results of this study are the first to demonstrate the benefit of PD-1 blockade in CLL patients with RT, and could change the landscape of therapy for RT patients if further validated. This trial was registered at www.clinicaltrials.gov as #NCT02332980.

Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

GATA-3 expression identifies a high-risk subset of PTCL, NOS with distinct molecular and clinical features.

The cell of origin and the tumor microenvironment's role remain elusive for the most common peripheral T-cell lymphomas (PTCLs). As macrophages promote the growth and survival of malignant T cells and are abundant constituents of the tumor microenvironment, their functional polarization was examined in T-cell lymphoproliferative disorders. Cytokines that are abundant within the tumor microenvironment, particularly interleukin (IL)-10, were observed to promote alternative macrophage polarization. Macrophage polarization was signal transducer and activator of transcription 3 dependent and was impaired by the Janus kinase inhibitor ruxolitinib. In conventional T cells, the production of T helper (Th)2-associated cytokines and IL-10, both of which promote alternative macrophage polarization, is regulated by the T-cell transcription factor GATA-binding protein 3 (GATA-3). Therefore, its role in the T-cell lymphomas was examined. GATA-3 expression was observed in 45% of PTCLs, not otherwise specified (PTCL, NOS) and was associated with distinct molecular features, including the production of Th2-associated cytokines. In addition, GATA-3 expression identified a subset of PTCL, NOS with distinct clinical features, including inferior progression-free and overall survival. Collectively, these data suggest that further understanding the cell of origin and lymphocyte ontogeny among the T-cell lymphomas may improve our understanding of the tumor microenvironment's pathogenic role in these aggressive lymphomas.

Anti-myeloma activity of Akt inhibition is linked to the activation status of PI3K/Akt and MEK/ERK pathway.

The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.

Expression of Myc, but not pSTAT3, is an adverse prognostic factor for diffuse large B-cell lymphoma treated with epratuzumab/R-CHOP.

STAT3 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.

Elevated serum IL-10 levels in diffuse large B-cell lymphoma: a mechanism of aberrant JAK2 activation.

Cytokines 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.

Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies.

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.

Sorafenib, a multikinase inhibitor, is effective in vitro against non-Hodgkin lymphoma and synergizes with the mTOR inhibitor rapamycin.

Non-Hodgkin lymphoma (NHL) represents a heterogenous group of neoplasias originating from lymphoid cells. Increased angiogenesis and expression of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR) have been found to be associated with NHL disease progression. Increase in VEGF and other cytokines stimulate signaling cascades, including the Ras/Raf/Mek/Erk pathway, resulting in increased proliferation and decreased apoptosis. Here, we report the in vitro antilymphoma activity of sorafenib, an inhibitor of VEGFR and Raf kinase. Sorafenib induced potent cytotoxicity in NHL cell lines and patient samples. This induction of cytotoxicity was associated with a corresponding increase in apoptotic cell death. Mechanism of action of sorafenib was investigated in follicular (DoHH2) and Burkitt lymphoma (Raji) cell lines. pStat3, pAkt, Mcl1, and Xiap were downregulated in both cell lines, whereas pErk decreased in Raji but not in DoHH2 cells following sorafenib treatment. IL6 was unable to prevent sorafenib induced repression of pStat3, pAkt, Mcl1, and Bcl-Xl. Sorafenib in combination with an mTORC1 inhibitor rapamycin demonstrated synergy in inducing cytotoxicity in NHL cells. Sorafenib/rapamycin combination resulted in downregulation of pAkt, pmTOR, p-p70S6K, p4EBP1, pGSK3ß, Mcl1, and Bcl-Xl. On the basis of our results, a clinical trial is underway using sorafenib with everolimus in NHL patients.