Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma are the most prevalent B-lymphocyte neoplasms in which abnormal activation of the Bruton tyrosine kinase (BTK)-mediated B-cell receptor signaling pathway contributes to pathogenesis. Ibrutinib is an oral covalent BTK inhibitor that has shown some efficacy in both indications. To improve ibrutinib efficacy through combination therapy, we first investigated differential gene expression in parental and ibrutinib-resistant cell lines to better understand the mechanisms of resistance. Ibrutinib-resistant TMD8 cells had higher BCL2 gene expression and increased sensitivity to ABT-199, a BCL-2 inhibitor. Consistently, clinical samples from ABC-DLBCL patients who experienced poorer response to ibrutinib had higher BCL2 gene expression. We further demonstrated synergistic growth suppression by ibrutinib and ABT-199 in multiple ABC-DLBCL, GCB-DLBCL, and follicular lymphoma cell lines. The combination of both drugs also reduced colony formation, increased apoptosis, and inhibited tumor growth in a TMD8 xenograft model. A synergistic combination effect was also found in ibrutinib-resistant cells generated by either genetic mutation or drug treatment. Together, these findings suggest a potential clinical benefit from ibrutinib and ABT-199 combination therapy. Mol Cancer Ther; 16(7); 1246-56. ©2017 AACR.

The role of PIM1 in the ibrutinib-resistant ABC subtype of diffuse large B-cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous lymphoma and the most common subtype of non-Hodgkin lymphoma, accounting for roughly 30% of newly diagnosed cases in the United States. DLBCL can be separated into the activated B cell-like (ABC) and germinal center B cell-like (GCB) subtypes, with distinct gene expression profiles, oncogenic aberrations, and clinical outcomes. ABC-DLBCL is characterized by chronically active B-cell receptor (BCR) signaling that can be modulated by Bruton's tyrosine kinase (BTK) activity. Thus, BTK serves as an attractive therapeutic target in this type of B-cell malignancy. Ibrutinib, a first-in-class, orally available covalent BTK inhibitor, has demonstrated clinical activity in several B-cell leukemias and lymphomas. A phase 1/2 clinical trial of single-agent ibrutinib in relapsed and refractory DLBCL patients revealed an overall response rate of 37% in ABC-DLBCL patients. However, responses to kinase-directed therapies are often limited by emerging resistance mechanisms that bypass the therapeutic target. Here we report the discovery of point mutations within the kinase PIM1 that reduce sensitivity to ibrutinib in ABC-DLBCL. These mutations stabilize PIM1 and affect upstream regulators and downstream targets of NF-κB signaling. The introduction of mutant PIM1 into an ABC-DLBCL cell line, TMD8, increased colony formation and decreased sensitivity to ibrutinib. In addition, ibrutinib-resistant cell lines generated by prolonged ibrutinib exposure in vitro upregulated PIM1 expression, consistent with a role for PIM1 in antagonizing ibrutinib activity. The combination of a pan-PIM inhibitor with ibrutinib synergistically inhibited proliferation in vitro and tumor growth in vivo. Together, these data provide a rationale for combining BTK and PIM1 inhibition in the treatment of ABC-DLBCL.

Decrease in brain soluble amyloid precursor protein ß (sAPPß) in Alzheimer's disease cortex.

Amyloid-ß peptide (Aß) is generated by sequential cleavage of the amyloid precursor protein (APP) by ß-site amyloid precursor protein cleaving enzyme 1 (ß-secretase, or BACE1) and γ-secretase. Several reports demonstrate increased BACE1 enzymatic activity in brain and cerebrospinal fluid (CSF) from Alzheimer's disease (AD) subjects, suggesting that an increase in BACE1-mediated cleavage of APP drives amyloid pathophysiology in AD. BACE1 cleavage of APP leads to the generation of a secreted N-terminal fragment of APP (sAPPß). To relate BACE1 activity better to endogenous APP processing in AD and control brains, we have directly measured brain sAPPß levels using a novel APP ß-site specific enzyme-linked immunosorbent assay. We demonstrate a significant reduction in brain cortical sAPPß levels in AD compared with control subjects. In the same brain samples, BACE1 activity was unchanged, full-length APP and sAPPα levels were significantly reduced, and Aß peptides were significantly elevated. In conclusion, a reduction in cortical brain sAPPß together with unchanged BACE1 activity suggests that this is due to reduced full-length APP substrate in late-stage AD subjects. These results highlight the need for multiparameter analysis of the amyloidogenic process to understand better AD pathophysiology in early vs. late-stage AD.

Molecular characterization of the effects of Y-27632.

Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extensions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.

Myelin-associated inhibitors regulate cofilin phosphorylation and neuronal inhibition through LIM kinase and Slingshot phosphatase.

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. We found that Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM (LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. We speculate that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors.

Neuronal responses to myelin are mediated by rho kinase.

CNS myelin inhibits axon growth due to the expression of several growth-inhibitory proteins, including myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein and Nogo. Myelin-associated inhibitory proteins activate rho GTPase in responsive neurons. Rho kinase (ROCK) has been implicated as a critical rho effector in this pathway due to the ability of the pharmacological inhibitor Y-27632 to circumvent myelin-dependent inhibition. Y-27632, however, inhibits the activity of additional kinases. Using three independent approaches, we provide direct evidence that ROCKII is activated in response to the myelin-associated inhibitor Nogo. We demonstrate that Nogo treatment enhances ROCKII translocation to the cellular membrane in PC12 cells and enhances ROCKII kinase activity towards an in vitro substrate. In addition, Nogo treatment enhances phosphorylation of myosin light chain II, a known ROCK substrate. Further, we demonstrate that primary dorsal root ganglia neurons can be rendered insensitive to the inhibitory effects of myelin via infection with dominant negative ROCK. Together these data provide direct evidence for a rho-ROCK-myosin light chain-II signaling cascade in response to myelin-associated inhibitors.