Persistent janus kinase-signaling in chronic lymphocytic leukemia patients on ibrutinib: Results of a phase I trial.

Methods to deepen clinical responses to ibrutinib are needed to improve outcomes for patients with chronic lymphocytic leukemia (CLL). This study aimed to determine the safety and efficacy of combining a janus kinase (JAK)-inhibitor with ibrutinib because JAK-mediated cytokine-signals support CLL cells and may not be inhibited by ibrutinib. The JAK1/2 inhibitor ruxolitinib was prescribed to 12 CLL patients with abnormal serum beta-2 microglobulin levels after 6 months or persistent lymphadenopathy or splenomegaly after 12 months on ibrutinib using a 3 + 3 phase 1 trial design (NCT02912754). Ibrutinib was continued at 420 mg daily and ruxolitinib was added at 5, 10, 15, or 20 mg BID for 3 weeks out of five for seven cycles. The break was mandated to avoid anemia and thrombocytopenia observed with ruxolitinib as a single agent in CLL. The combination was well-tolerated without dose-limiting toxicities. Cyclic changes in platelets, lymphocytes, and associated chemokines and thrombopoietic factors were observed and partial response criteria were met in 2 of 12 patients. The results suggest that JAK-signaling helps CLL cells persist in the presence of ibrutinib and ruxolitinib with ibrutinib is well-tolerated and may be a useful regiment to use in combination therapies for CLL.

Ibrutinib reprograms the glucocorticoid receptor in chronic lymphocytic leukemia cells.

Glucocorticoid (GC) receptor (GR) phosphorylation and signature genes were studied in chronic lymphocytic leukemia (CLL) cells to help place GCs within modern treatment algorithms. In contrast to normal B and T cells, transcription of GC-regulated genes was not rhythmic and the synthetic GC dexamethasone (DEX) could not inhibit toll-like receptor (TLR)-responses in CLL cells. This intrinsic GC-resistance was associated with aberrant GR-phosphorylation on activating Ser211 and inhibitory Ser226 sites. Ibrutinib increased transcription of the GR-signature gene GILZ in circulating CLL cells along with GR(pS211)/GR(pS226) ratios and lytic sensitivity to DEX that were not reversed by the competitive antagonist mifepristone in vitro. However, ibrutinib could not improve GR-responses in circulating CLL cells activated with IL2 and TLR7/8 agonists to mimic conditions in pseudofollicle microenvironments. Addition of the janus kinase inhibitor ruxolitinib to block ibrutinib-insensitive signals increased GILZ transcription in pseudofollicle conditions in vitro and in a clinical trial (NCT02912754), and also increased GR(S211)/GR(S226) ratios and DEX-mediated killing in patient samples in vitro. These observations suggest that intrinsic resistance to endogenous GCs is characteristic of CLL cells and ibrutinib may help increase the therapeutic activity of GCs by non-canonical activation of GR.

Targeting the IL-17/IL-6 axis can alter growth of Chronic Lymphocytic Leukemia in vivo/in vitro.

The tumor microenvironment (TME) is critical to the longevity of tumor B cells in chronic lymphocytic leukemia (CLL). Bone marrow mesenchymal stem cells (BMMSCs) and the cytokines they produce including IL-6 are important components of the TME in CLL. We found BMMSCs supported the survival of CLL cells in vitro through an IL-6 dependent mechanism. IL-17 which induces IL-6 generation in a variety of cells increased production of IL-6 both in CLL cells and BMMSCs in vitro. In a xenograft CLL mouse model, BMMSCs and the culture supernatant of BMMSCs increased engraftment of CLL cells through an IL-6 mediated mechanism with human recombinant IL-6 showing similar effects in vivo. Human recombinant IL-17 treatment also increased CLL engraftment in mice through an IL-6 mediated mechanism. Plasma of CLL patients showed elevated levels of both IL-6 and IL-17 by ELISA compared with healthy controls, with levels of IL-6 linearly correlated with IL-17 levels. CLL patients requiring fludarabine based chemotherapy expressed higher levels of IL-6 and IL-17, while CLL patients with the lowest levels of IgA/IgM had higher levels of IL-6, but not IL-17. These data imply an important role for the IL-17/IL-6 axis in CLL which could be therapeutic targets.

Low Density Lipoproteins Amplify Cytokine-signaling in Chronic Lymphocytic Leukemia Cells.

Recent studies suggest there is a high incidence of elevated low-density lipoprotein (LDL) levels in Chronic Lymphocytic Leukemia (CLL) patients and a survival benefit from cholesterol-lowering statin drugs. The mechanisms of these observations and the kinds of patients they apply to are unclear. Using an in vitro model of the pseudofollicles where CLL cells originate, LDLs were found to increase plasma membrane cholesterol, signaling molecules such as tyrosine-phosphorylated STAT3, and activated CLL cell numbers. The signaling effects of LDLs were not seen in normal lymphocytes or glycolytic lymphoma cell-lines but were restored by transduction with the nuclear receptor PPARδ, which mediates metabolic activity in CLL cells. Breakdown of LDLs in lysosomes was required for the amplification effect, which correlated with down-regulation of HMGCR expression and long lymphocyte doubling times (LDTs) of 53.6±10.4months. Cholesterol content of circulating CLL cells correlated directly with blood LDL levels in a subgroup of patients. These observations suggest LDLs may enhance proliferative responses of CLL cells to inflammatory signals. Prospective clinical trials are needed to confirm the therapeutic potential of lowering LDL concentrations in CLL, particularly in patients with indolent disease in the "watch-and-wait" phase of management.

Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells.

Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors.

Microenvironmental interleukin-6 suppresses toll-like receptor signaling in human leukemia cells through miR-17/19A.

The regulation of toll-like receptor (TLR) signaling in a tumor microenvironment is poorly understood despite its importance in cancer biology. To address this problem, TLR7-responses of chronic lymphocytic leukemia (CLL) cells were studied in the presence and absence of a human stromal cell-line derived from a leukemic spleen. CLL cells alone produced high levels of tumor necrosis factor (TNF)-α and proliferated in response to TLR7-agonists. A signal transducer and activator of transcription 3 -activating stromal factor, identified as interleukin (IL)-6, was found to upregulate microRNA (miR)-17 and miR-19a, target TLR7 and TNFA messenger RNA, and induce a state of tolerance to TLR7-agonists in CLL cells. Overexpression of the miR-17-92 cluster tolerized CLL cells directly and miR-17 and miR-19a antagomiRs restored TLR7-signaling. Inhibition of IL-6 signaling with antibodies or small-molecule Janus kinase inhibitors reversed tolerization and increased TLR7-stimulated CLL cell numbers in vitro and in NOD-SCIDγc (null) mice. These results suggest IL-6 can act as tumor suppressor in CLL by inhibiting TLR-signaling.

Resveratrol has anti-leukemic activity associated with decreased O-GlcNAcylated proteins.

CLL cells are characterized by high levels of proteins that are post-translationally modified by O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties, but it is not clear whether O-GlcNAc is a relevant therapeutic target. The neutraceutical resveratrol is cytotoxic to chronic lymphocytic leukemia cells in vitro. In this study, we found that resveratrol has therapeutic activity as a single agent in vivo in both human chronic lymphocytic leukemia patients and mice with erythroleukemia. Blood and splenic O-GlcNAc levels reflected the changes in tumor burden. Resveratrol directly lowered O-GlcNAc levels in leukemia cells through proteasomal activation, but increasing O-GlcNAc levels in vitro did not prevent cell death. These findings suggest that resveratrol has potential as a novel treatment for some forms of chronic and acute leukemia, and the measurement of O-GlcNAc levels could be a surrogate marker for therapeutic responses.