The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells

Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. U266-resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells. Elimination of velcade-induced poly-ubiquitinated proteins and protein aggregates was drastically stimulated in the resistant cells and correlated with increased cell survival. Inhibition of the lysosomal activity in velcade-resistant cells resulted in an increase of cell aggregates and decrease survival, indicating that aggregates are eliminated through lysosomal degradation. In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival

Mechanisms of AXL overexpression and function in Imatinib-resistant chronic myeloid leukemia cells

AXL is a receptor tyrosine kinase of the TAM family, the function of which is poorly understood. We previously identified AXL overexpression in Imatinib (IM)-resistant CML cell lines and patients. The present study was conducted to investigate the role of AXL and the mechanisms underlying AXL overexpression in Tyrosine Kinase Inhibitor (TKI)-resistant CML cells. We present evidence that high AXL expression level is a feature of TKI-resistant CML cells and knockdown of AXL sensitized TKI-resistant cells to IM. In addition, expression of wild-type AXL but not a dominant negative form of AXL confers IM-sensitive CML cells the capacity to resist IM effect. AXL overexpression required PKCα and β and constitutive activation of ERK1/2. Accordingly, GF109203X a PKC inhibitor, U0126 a MEK1 inhibitor and PKCα/β knockdown restore sensitivity to IM while PKCα or PKCβ overexpression in CML cells promotes protection against IM-induced cell death. Finally, using luciferase promoter activity assays we established that AXL is regulated transcriptionally through the AP1 transcription factor. Our findings reveal an unexpected role of AXL in resistance to TKI in CML cells, identify the molecular mechanisms involved in its overexpression and support the notion that AXL is a new marker of resistance to TKI in CML

BCL2L10 is a predictive factor for resistance to Azacitidine in MDS and AML patients

Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients

Escherichia coli α-Hemolysin Counteracts the Anti-Virulence Innate Immune Response Triggered by the Rho GTPase Activating Toxin CNF1 during Bacteremia

International audienceThe detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1 beta signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1 beta antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1 beta and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the Phi-hemolysin toxin inhibits IL-1 beta secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia

Implication and Regulation of AMPK during Physiological and Pathological Myeloid Differentiation

AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase consisting of the arrangement of various α β, and γ isoforms that are expressed differently depending on the tissue or the cell lineage. AMPK is one of the major sensors of energy status in mammalian cells and as such plays essential roles in the regulation of cellular homeostasis, metabolism, cell growth, differentiation, apoptosis, and autophagy. AMPK is activated by two upstream kinases, the tumor suppressor liver kinase B1 (LKB1) and the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) through phosphorylation of the kinase on Thr172, leading to its activation. In addition, AMPK inhibits the mTOR pathway through phosphorylation and activation of tuberous sclerosis protein 2 (TSC2) and causes direct activation of unc-51-like autophagy activating kinase 1 (ULK1) via phosphorylation of Ser555, thus promoting initiation of autophagy. Although it is well established that AMPK can control the differentiation of different cell lineages, including hematopoietic stem cells (HSCs), progenitors, and mature hematopoietic cells, the role of AMPK regarding myeloid cell differentiation is less documented. The differentiation of monocytes into macrophages triggered by colony stimulating factor 1 (CSF-1), a process during which both caspase activation (independently of apoptosis induction) and AMPK-dependent stimulation of autophagy are necessary, is one noticeable example of the involvement of AMPK in the physiological differentiation of myeloid cells. The present review focuses on the role of AMPK in the regulation of the physiological and pathological differentiation of myeloid cells. The mechanisms of autophagy induction by AMPK will also be addressed, as autophagy has been shown to be important for differentiation of hematopoietic cells. In addition, myeloid malignancies (myeloid leukemia or dysplasia) are characterized by profound defects in the establishment of proper differentiation programs. Reinduction of a normal differentiation process in myeloid malignancies has thus emerged as a valuable and promising therapeutic strategy. As AMPK seems to exert a key role in the differentiation of myeloid cells, notably through induction of autophagy, we will also discuss the potential to target this pathway as a pro-differentiating and anti-leukemic strategy in myeloid malignancies