The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells.

Previous studies demonstrated that imatinib mesylate (IM) induces autophagy in chronic myeloid leukemia (CML) and that this process is critical to cell survival upon therapy. However, it is not known if the autophagic process differs at basal levels between CML patients and healthy individuals and if pretreatment CML cells harbor unique autophagy characteristics that could predict patients' clinical outcomes. We now demonstrate that several key autophagy genes are differentially expressed in CD34(+) hematopoietic stem/progenitor cells, with the highest transcript levels detected for ATG4B, and that the transcript and protein expression levels of ATG4 family members, ATG5 and BECLIN-1 are significantly increased in CD34(+) cells from chronic-phase CML patients (P < .05). Importantly, ATG4B is differentially expressed in pretreatment CML stem/progenitor cells from subsequent IM responders vs IM nonresponders (P < .05). Knockdown of ATG4B suppresses autophagy, impairs the survival of CML stem/progenitor cells and sensitizes them to IM treatment. Moreover, deregulated expression of ATG4B in CD34(+) CML cells inversely correlates with transcript levels of miR-34a, and ATG4B is shown to be a direct target of miR-34a. This study identifies ATG4B as a potential biomarker for predicting therapeutic response in treatment-naïve CML stem/progenitor cells and uncovers ATG4B as a possible drug target in these cells.

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells.

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.

Rap GTPase-mediated adhesion and migration: A target for limiting the dissemination of B-cell lymphomas?

B-cell lymphomas, which arise in lymphoid organs, can spread rapidly via the circulatory system and form solid tumors within multiple organs. Rate-limiting steps in this metastatic process may be the adhesion of lymphoma cells to vascular endothelial cells, their exit from the vasculature and their migration to tissue sites that will support tumor growth. Thus proteins that control B cell adhesion and migration are likely to be key factors in lymphoma dissemination, and hence potential targets for therapeutic intervention. The Rap GTPases are master regulators of integrin activation, cell motility and the underlying cytoskeletal, adhesion and membrane dynamics. We have recently shown that Rap activation is critical for B-lymphoma cells to undergo transendothelial migration in vitro and in vivo. As a consequence, suppressing Rap activation impairs the ability of intravenously injected B-lymphoma cells to form solid tumors in the liver and other organs. We discuss this work in the context of targeting Rap, its downstream effectors, or other regulators of B cell adhesion and migration as an approach for limiting the dissemination of B-lymphoma cells and the development of secondary tumors.

The rap GTPases regulate B cell morphology, immune-synapse formation, and signaling by particulate B cell receptor ligands.

B lymphocytes spread and extend membrane processes when searching for antigens and form immune synapses upon contacting cells that display antigens on their surface. Although these dynamic morphological changes facilitate B cell activation, the signaling pathways underlying these processes are not fully understood. We found that activation of the Rap GTPases was essential for these changes in B cell morphology. Rap activation was important for B cell receptor (BCR)- and lymphocyte-function-associated antigen-1 (LFA-1)-induced spreading, for BCR-induced immune-synapse formation, and for particulate BCR ligands to induce localized F-actin assembly and membrane-process extension. Rap activation and F-actin assembly were also required for optimal BCR signaling in response to particulate antigens but not soluble antigens. Thus by controlling B cell morphology and cytoskeletal organization, Rap might play a key role in the activation of B cells by particulate and cell-associated antigens.

Activation of the Rap GTPases in B lymphocytes modulates B cell antigen receptor-induced activation of Akt but has no effect on MAPK activation.

Signaling by the B cell antigen receptor (BCR) activates the Rap1 and Rap2 GTPases, putative antagonists of Ras-mediated signaling. Because Ras can activate the Raf-1/ERK pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we asked whether Rap activation limits the ability of the BCR to signal via these pathways. To do this, we blocked the activation of endogenous Rap1 and Rap2 by expressing the Rap-specific GTPase-activating protein RapGAPII. Preventing Rap activation had no effect on BCR-induced activation of ERK. In contrast, BCR-induced phosphorylation of Akt on critical activating sites was increased 2- to 3-fold when Rap activation was blocked. Preventing Rap activation also increased the ability of the BCR to stimulate Akt-dependent phosphorylation of the FKHR transcription factor on negative regulatory sites and decreased the levels of p27Kip1, a pro-apoptotic factor whose transcription is enhanced by FKHR. Moreover, preventing Rap activation reduced BCR-induced cell death in the WEHI-231 B cell line. Thus activation of endogenous Rap by the BCR limits BCR-induced activation of the PI3K/Akt pathway, opposes the subsequent inhibition of the FKHR/p27Kip1 pro-apoptotic module, and enhances BCR-induced cell death. Consistent with the idea that Rap-GTP is a negative regulator of the PI3K/Akt pathway, expressing constitutively active Rap2 (Rap2V12) reduced BCR-induced phosphorylation of Akt and FKHR. Finally, our finding that Rap2V12 can bind PI3K and inhibit its activity in a manner that depends upon BCR engagement provides a potential mechanism by which Rap-GTP limits activation of the PI3K/Akt pathway, a central regulator of B cell growth and survival.