Study of Rho GTPases in multiple myeloma: involvement of RhoU in disease initiation and progression

Multiple myeloma (MM) is a cancer of post-germinal center B cells characterized by a clonal proliferation of long-lived plasma cells inside the bone marrow. MM cells typically exhibit numerous structural and numerical chromosomal aberrations besides the presence of mutations in oncogenes and tumor-suppressor genes. Recently, a lot of attention has been drawn towards the tumor microenvironment. The interaction between malignant plasma cells and other cells inside the bone marrow is thought to be essential for the survival and expansion of MM. Indeed, stromal cells are able to produce growth factors that are important in sustaining the proliferation of MM cells, for example, interleukin-6 (IL-6). IL-6 triggers the signal downstream of its receptor IL-6R, leading to the activation of the JAK/STAT pathway. An important target of this pathway is STAT3 transcription factor. STAT3 binds to the promotor region of a set of genes that regulate cell growth, proliferation, survival, mitosis, adhesion/migration, and are extremely important in controlling the inflammatory response. RhoU is an atypical member of the Rho GTPase family that lies downstream of STAT3 activation. This GTPase is constitutively active whenever expressed and could mediate the effects of STAT3 in regulating the cytoskeleton dynamics. In MM nothing is known about this G protein. Here we demonstrate for the first time a role for RhoU in regulating the F-actin cytoskeleton of MM cells. RhoU was found heterogeneously expressed in MM patients’ cells, significantly modulated with disease progression, and expressed at higher levels in patients with bad prognosis mutations including t(4;14), del(13) and 1q gain. Different levels of RhoU mRNA correlate with a diverse gene expression profile in 557 genes. We have also found that it significantly clusters with cell cycle and DNA damage genes. Importantly, its expression positively correlates with cyclin D2 expression, and negatively correlates with the expression of cell cycle control and DNA damage response genes. In MM cell lines, RhoU is over-expressed in IL-6 dependent cell lines, while its expression is down-modulated in those that can proliferate independently of IL-6 stimulus. MM cell lines were able to up-regulate RhoU mRNA expression in response to IL-6 stimulus through the activation of STAT3. RhoU silencing led to an accumulation of actin stress fibers, an increase in adhesion and a blockade in migration. Lastly, immunomodulatory drugs (IMIDs) were recently found to control the activation of classical GTPases like Cdc42 and RhoA. In accordance with this, Lenalidomide positively regulated STAT3 activation leading to an increase in RhoU expression that resulted in a higher migration capability of MM cell lines, indicating that IMIDs can also alter the expression of atypical GTPases

Prosurvival autophagy is regulated by protein kinase CK1 alpha in multiple myeloma

Multiple myeloma (MM) is a tumor of plasma cells (PCs). Due to the intense immunoglobulin secretion, PCs are prone to endoplasmic reticulum stress and activate several stress-managing pathways, including autophagy. Indeed, autophagy deregulation is maladaptive for MM cells, resulting in cell death. CK1α, a pro-survival kinase in MM, has recently been involved as a regulator of the autophagic flux and of the transcriptional competence of the autophagy-related transcription factor FOXO3a in several cancers. In this study, we investigated the role of CK1α in autophagy in MM. To study the autophagic flux we generated clones of MM cell lines expressing the mCherry-eGFP-LC3B fusion protein. We observed that CK1 inhibition with the chemical ATP-competitive CK1 α/δ inhibitor D4476 resulted in an impaired autophagic flux, likely due to an alteration of lysosomes acidification. However, D4476 caused the accumulation of the transcription factor FOXO3a in the nucleus, and this was paralleled by the upregulation of mRNA coding for autophagic genes. Surprisingly, silencing of CK1α by RNA interference triggered the autophagic flux. However, FOXO3a did not shuttle into the nucleus and the transcription of autophagy-related FOXO3a-dependent genes was not observed. Thus, while the chemical inhibition with the dual CK1α/δ inhibitor D4476 induced cell death as a consequence of an accumulation of ineffective autophagic vesicles, on the opposite, CK1α silencing, although it also determined apoptosis, triggered a full activation of the early autophagic flux, which was then not supported by the upregulation of autophagic genes. Taken together, our results indicate that the family of CK1 kinases may profoundly influence MM cells survival also through the modulation of the autophagic pathway

Study of Rho GTPases in multiple myeloma: involvement of RhoU in disease initiation and progression

Multiple myeloma (MM) is a cancer of post-germinal center B cells characterized by a clonal proliferation of long-lived plasma cells inside the bone marrow. MM cells typically exhibit numerous structural and numerical chromosomal aberrations besides the presence of mutations in oncogenes and tumor-suppressor genes. Recently, a lot of attention has been drawn towards the tumor microenvironment. The interaction between malignant plasma cells and other cells inside the bone marrow is thought to be essential for the survival and expansion of MM. Indeed, stromal cells are able to produce growth factors that are important in sustaining the proliferation of MM cells, for example, interleukin-6 (IL-6). IL-6 triggers the signal downstream of its receptor IL-6R, leading to the activation of the JAK/STAT pathway. An important target of this pathway is STAT3 transcription factor. STAT3 binds to the promotor region of a set of genes that regulate cell growth, proliferation, survival, mitosis, adhesion/migration, and are extremely important in controlling the inflammatory response. RhoU is an atypical member of the Rho GTPase family that lies downstream of STAT3 activation. This GTPase is constitutively active whenever expressed and could mediate the effects of STAT3 in regulating the cytoskeleton dynamics. In MM nothing is known about this G protein. Here we demonstrate for the first time a role for RhoU in regulating the F-actin cytoskeleton of MM cells. RhoU was found heterogeneously expressed in MM patients’ cells, significantly modulated with disease progression, and expressed at higher levels in patients with bad prognosis mutations including t(4;14), del(13) and 1q gain. Different levels of RhoU mRNA correlate with a diverse gene expression profile in 557 genes. We have also found that it significantly clusters with cell cycle and DNA damage genes. Importantly, its expression positively correlates with cyclin D2 expression, and negatively correlates with the expression of cell cycle control and DNA damage response genes. In MM cell lines, RhoU is over-expressed in IL-6 dependent cell lines, while its expression is down-modulated in those that can proliferate independently of IL-6 stimulus. MM cell lines were able to up-regulate RhoU mRNA expression in response to IL-6 stimulus through the activation of STAT3. RhoU silencing led to an accumulation of actin stress fibers, an increase in adhesion and a blockade in migration. Lastly, immunomodulatory drugs (IMIDs) were recently found to control the activation of classical GTPases like Cdc42 and RhoA. In accordance with this, Lenalidomide positively regulated STAT3 activation leading to an increase in RhoU expression that resulted in a higher migration capability of MM cell lines, indicating that IMIDs can also alter the expression of atypical GTPases.Il mieloma multiplo (MM) è un tumore caratterizzato dalla proliferazione di plasmacellule clonali all'interno del midollo osseo. Tipicamente, le cellule di MM presentano numerose aberrazioni cromosomiche strutturali e numeriche, oltre alla presenza di mutazioni in oncogeni e geni oncosoppressori. Recentemente molta attenzione è stata posta nei confronti del microambiente tumorale. L'interazione all'interno del midollo osseo tra le plasmacellule maligne e altre cellule è essenziale per la sopravvivenza e l'espansione del MM. Infatti, le cellule stromali sono in grado di produrre fattori di crescita importanti nel sostenere la proliferazione delle cellule di MM, per esempio, l'interleuchina-6 (IL-6). IL-6 attiva il segnale a valle del suo recettore IL-6R, determinando l'attivazione del pathway di JAK/STAT. Un importante bersaglio di questa cascata di segnale è il fattore di trascrizione STAT3. STAT3 si lega al promotore di una serie di geni che regolano la crescita cellulare, la proliferazione, la sopravvivenza, la mitosi, l'adesione e la migrazione e che ricoprono un ruolo di estrema importanza nel controllo della risposta infiammatoria. RhoU è un membro atipico della famiglia delle Rho GTPasi che si trova a valle dell'attivazione di STAT3. Questa GTPasi è costitutivamente attiva quando espressa ed è sospettata di mediare gli effetti di STAT3 nella regolazione delle dinamiche del citoscheletro. Il ruolo di questa proteina nel MM è ignoto. In questo lavoro si dimostra per la prima volta un ruolo di RhoU nella regolazione del citoscheletro di actina delle cellule di MM. RhoU è stato riscontrato essere eterogeneamente espresso in cellule di pazienti di MM, significativamente modulato con la progressione della malattia ed espresso a livelli più alti in pazienti con aberrazioni t(4; 14), del(13) e 1q gain. Diversi livelli di RhoU correlano con un diverso profilo di espressione genica in 557 geni. È importante sottolineare anche che l'espressione di RhoU correla positivamente con l'espressione della ciclina D2 e negativamente con l'espressione di geni che controllano il ciclo cellulare e la risposta a danni al DNA. In linee cellulari di MM, RhoU è sovraespresso in quelle IL-6-dipendenti, mentre la sua espressione è down-modulata in quelle che possono proliferare indipendentemente da IL-6. Le linee cellulari di MM sono in grado di up-regolare l'espressione RhoU in risposta allo stimolo di IL-6, attraverso l'attivazione di STAT3. Il silenziamento di RhoU porta ad un accumulo di fibre di actina, un aumento della adesione e un blocco nella migrazione. Infine, è stato recentemente dimostrato come i farmaci immunomodulatori (IMiDs) controllino l'attivazione delle GTPasi classiche come Cdc42 e RhoA. In accordo con questo, abbiamo svelato che Lenalidomide regola positivamente l'attivazione di STAT3 portando ad un aumento dell'espressione di RhoU. Questo porta ad una capacità di migrazione più elevata nelle linee cellulari di MM, indicando che gli IMiD sono anche in grado di alterare l'espressione di GTPasi atipiche

CX-4945, a selective inhibitor of casein kinase 2, synergizes with B cell receptor signaling inhibitors in inducing diffuse large B cell lymphoma cell death

Approximately one third of Diffuse Large B cell Lymphomas (DLBCL) are refractory or relapse. Novel therapeutic approaches under scrutiny include inhibitors of B-cell receptor (BCR) signaling. Protein kinase CK2 propels survival, proliferation and stress response in solid and hematologic malignancies and promotes a "non-oncogene addiction" phenotype. Whether this kinase regulates BCR signaling thus being a suitable pharmacological target in DLBCL is unknown

The small GTPase RhoU lays downstream of JAK/STAT signaling and mediates cell migration in multiple myeloma

Multiple myeloma is a post-germinal center B-cell neoplasm, characterized by the proliferation of malignant bone marrow plasma cells, whose survival and proliferation is sustained by growth factors and cytokines present in the bone marrow microenvironment. Among them, IL-6 triggers the signal downstream of its receptor, leading to the activation of the JAK/STAT pathway. The atypical GTPase RhoU lays downstream of STAT3 transcription factor and could be responsible for mediating its effects on cytoskeleton dynamics. Here we demonstrate that RHOU is heterogeneously expressed in primary multiple myeloma cells and significantly modulated with disease progression. At the mRNA level, RHOU expression in myeloma patients correlated with the expression of STAT3 and its targets MIR21 and SOCS3. Also, IL-6 stimulation of human myeloma cell lines up-regulated RHOU through STAT3 activation. On the other hand, RhoU silencing led to a decrease in cell migration with the accumulation of actin stress fibers, together with a decrease in cyclin D2 expression and in cell cycle progression. Furthermore, we found that even though lenalidomide positively regulated RhoU expression leading to higher cell migration rates, it actually led to cell cycle arrest probably through a p21 dependent mechanism. Lenalidomide treatment in combination with RhoU silencing determined a loss of cytoskeletal organization inhibiting cell migration, and a further increase in the percentage of cells in a resting phase. These results unravel a role for RhoU not only in regulating the migratory features of malignant plasma cells, but also in controlling cell cycle progression

Inactivation of CK1\u3b1 in multiple myeloma empowers drug cytotoxicity by affecting AKT and f-catenin survival signaling pathways

Recent evidence indicates that protein kinase CK1\u3b1 may support the growth of multiple myeloma (MM) plasma cells. Here, by analyzing a large cohort of MM cases, we found that high CK1\u3b1 mRNA levels are virtually associated with all MM patients. Moreover, we provided functional evidence that CK1\u3b1 activity is essential for malignant plasma cell survival even in the protective niche generated by co-cultures with bone marrow stromal cells. We demonstrated that CK1\u3b1 inactivation, while toxic for myeloma cells, is dispensable for the survival of healthy B lymphocytes and stromal cells. Disruption of CK1\u3b1 function in myeloma cells resulted in decreased Mdm2, increased p53 and p21 and reduced expression of \u3b2-catenin and AKT. These effects were mediated partially by p53 and caspase activity. Finally, we discovered that CK1\u3b1 inactivation enhanced the cytotoxic effect of both bortezomib and lenalidomide. Overall, our study supports a role for CK1\u3b1 as a potential therapeutic target in MM in combination with proteasome inhibitors and/or immunomodulatory drugs

CK2β-regulated signaling controls B cell differentiation and function

frequency in CK2b KO mice suggesting the importance of the b subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation

CK2β-regulated signaling controls B cell differentiation and function

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the beta regulatory subunit of CK2. CK2 beta(KO) mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2 beta(KO) animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2 beta(KO) mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2 beta have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2 beta(KO) mice suggesting the importance of the beta subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation

CK2β Regulates Hematopoietic Stem Cell Biology and Erythropoiesis

The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the β regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2β knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2β were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis