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

Elotuzumab plus pomalidomide and dexamethasone in relapsed/refractory multiple myeloma: a multicenter, retrospective real-world experience with 200 cases outside of controlled clinical trials

In the ELOQUENT-3 trial, the combination of elotuzumab, pomalidomide and dexamethasone (EloPd) proved a superior clinical benefit over Pd with a manageable toxicity profile, leading to its approval in relapsed/refractory multiple myeloma (RRMM), who had received at least two prior therapies, including lenalidomide and a proteasome inhibitor (PI). We report here a real-world experience of 200 RRMMs treated with EloPd in 35 Italian centers outside of clinical trials. In our dataset, the median number of prior lines of therapy was 2, with 51% of cases undergoing autologous stem cell transplant (ASCT) and 73% exposed to daratumumab. After a median follow-up of 9 months, 126 patients stopped EloPd, most of them (88.9%) because of disease progression. The overall response rate (ORR) was 55.4%, in line with the pivotal trial results. Regarding adverse events, our cohort experienced a toxicity profile similar to the ELOQUENT-3 trial, with no significant differences between younger (<70 years) and older patients. The median progression-free survival (PFS) was 7 months, shorter than that observed in the ELOQUENT-3, probably due to the different clinical characteristics of the two cohorts. Interestingly, the ISS stage III (HR:2.55) was associated with worse PFS. Finally, our series's median overall survival (OS) was shorter than that observed in the ELOQUENT-3 trial (17.5 versus 29.8 months). In conclusion, our real-world study confirms EloPd as a safe and possible therapeutic choice for RRMM who received at least two prior therapies, including lenalidomide and a PI

Not all LGL leukemias are created equal

: Large Granular Lymphocyte (LGL) Leukemia is a rare, heterogeneous even more that once thought, chronic lymphoproliferative disorder characterized by the clonal expansion of T- or NK-LGLs that requires appropriate immunophenotypic and molecular characterization. As in many other hematological conditions, genomic features are taking research efforts one step further and are also becoming instrumental in refining discrete subsets of LGL disorders. In particular, STAT3 and STAT5B mutations may be harbored in leukemic cells and their presence has been linked to diagnosis of LGL disorders. On clinical grounds, a correlation has been established in CD8+ T-LGLL patients between STAT3 mutations and clinical features, in particular neutropenia that favors the onset of severe infections. Revisiting biological aspects, clinical features as well as current and predictable emerging treatments of these disorders, we will herein discuss why appropriate dissection of different disease variants is needed to better manage patients with LGL disorders

Large granular lymphocyte leukemia

Large granular lymphocyte (LGL) disorders account for several conditions characterized by the proliferation of clonal LGLs with cytotoxic activity. According to the LGL immunophenotype, most cases represent expansions of TCRα/β+ LGL, displaying a CD8+ CD4− or less frequently a CD4+ CD8−/+dim phenotype with variable expression of cytotoxic NK cell antigens, including CD57, CD16, and CD56. Clonal expansions of TCRγ/δ+ LGL are also described. Proliferations of CD3− CD16+ NK cells with a restricted pattern of NK receptors usually comprise 15% of the total. Morphologic, immunophenotypic and molecular analyses are mandatory for a correct diagnosis of disease. The JAK/STAT pathway is typically involved in sustaining LGL proliferation and recently the presence of STAT3 and STAT5B somatic mutations has been reported as a hallmark of the disease and included in the recent 2017 WHO classification. Understanding how the leukemic LGL clone survives is central to reconcile the molecular mechanisms to the clinical behavior of disease, in order to find new clues to overcome the still unacceptable limit of the conventional immunosuppressive treatments

Safety of Rapid Daratumumab Infusion: A Retrospective, Multicenter, Real-Life Analysis on 134 Patients With Multiple Myeloma

BackgroundThe anti-CD38 monoclonal antibody daratumumab is the backbone of most anti-multiple myeloma (MM) regimens. To mitigate the risk of infusion-related reactions (IRRs), intravenous daratumumab administration requires 7 hours for the first infusion and 3.5-4 hours thereafter, thus making daratumumab-containing regimens burdensome for patients and health care resources. Preliminary data suggest that a rapid (90-minute) infusion of daratumumab is safe and does not increase IRRs. The rapid schedule was adopted by our centers since 2019. MethodsWe conducted an observational multi-center, real-life study to assess the safety of rapid daratumumab infusion protocol from the third administration in relapsed MM patients receiving daratumumab alone or in combination with lenalidomide-dexamethasone or bortezomib-dexamethasone. The primary endpoint was the safety of the rapid infusion protocol, particularly in terms of IRRs. ResultsA total of 134 MM patients were enrolled. IRRs occurred in 7 (5%) patients and were mostly mild (6/7 of grade 1-2), with only 1 patient experiencing a grade 3 IRR. Due to the IRRs, 5 (3.7%) patients discontinued the rapid infusions and resumed daratumumab at the standard infusion rate, while 1 patient permanently discontinued daratumumab. In 4/7 patients (57%), IRRs occurred while resuming rapid daratumumab infusions after a temporary interruption (2-4 months). No other adverse event was considered related to the rapid infusion protocol. ConclusionsOur findings confirmed the safety of rapid daratumumab infusions starting from the third administration. In case of prolonged daratumumab interruption, it is advisable to resume infusions at the standard rate (3.5 hours) before switching to the rapid infusion

Identification of a STAT3-miRNA Axis in T-LGL Leukemia

Introduction T large granular lymphocytes leukemia (T-LGLL) is a rare disease characterized by the abnormal expansion of T-large granular lymphocytes (T-LGLs) in the peripheral blood. The etiology of this disease is still largely unknown. LGL proliferation is maintained through an impairment of the apoptotic machinery due to the activation of many survival signals. Among these, JAK/STAT signaling represents one of the most important deregulated pathways in T-LGLL. In particular, leukemic LGLs are equipped with STAT3 constitutively over-expressed and over-activated. Moreover, in 30-40% of patients, STAT3 has been demonstrated carrying hot-spot mutations, likely resulting in STAT3 activation. Although STAT3 is an inducer of transcription of a large number of oncogenes, its relationship with microRNAs (miRNAs) has not yet been extensively evaluated in T-LGLL patients. As a matter of fact, several miRNAs contribute to normal hematopoietic processes and many miRNAs act both as tumor suppressors and oncogenes in the pathology of hematological malignancies, including acute and chronic leukemias and lymphomas, where they contribute to lymphomagenesis acting in various cellular functions, such as the regulation of cell survival and proliferation. Aims We investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. A high throughput quantitative and qualitative analysis of the miRNA expression profile in leukemic LGLs compared to healthy controls was performed with the aim to investigate whether STAT3 activation and/or mutation were correlated to some miRNAs in leukemic LGLs. Methods Six patients (3 characterized by STAT3 mutations and 3 with wild type STAT3) and three healthy controls were enrolled in a pilot study. STAT3 mRNA expression and protein activation levels were analyzed by Real Time-PCR and Western Blot, respectively. The expression level of 756 mature miRNAs was assessed by using a TaqMan-based Low Density Array on purified LGLs. Experimental data were analyzed by ViiA7 RUO software and the relative miRNA expression values were calculated using U6 as endogenous control. miRNA array data underwent hierarchical cluster analysis (HCL) by using MEV. miRNAs with a 2 or 0.5 fold change and p value < 0.05 in samples as compared to controls were considered as differentially expressed. Results of this pilot study were validated on additional 12 T-LGLL and 3 healthy controls subjects. Results Two clusters were identified by HCL analysis: cluster A included healthy controls and LGL patients characterized by comparably low levels of STAT3 activation (S3low) and absence of STAT3 mutations. Cluster B included four patients characterized by high levels of STAT3 activation (S3high). Remarkably, three out of four LGL patients in cluster B shared STAT3 mutation. Comparative analysis of the miRNAs expressed identified 33 miRNAs upregulated and 9 miRNAs downregulated in S3high as compared to S3low. Interestingly, the level of expression of these selected miRNAs correlated with the level of STAT3 expression/activation in LGL. Among these, three miRNAs, miR-484, miR-501 and miR-1249, have been validated and confirmed in the validation cohort. Conclusions These data firstly describe the miRNA pattern in T-LGLL, providing evidence that a series of miRNAs are correlated with relevant key factors in T-LGLL pathogenesis, including STAT3 activation/expression and mutations. Our results suggest the hypothesis that STAT3 could mediate its role through some defined miRNAs

Defining TCRγδ lymphoproliferative disorders by combined immunophenotypic and molecular evaluation

Tγδ large granular lymphocyte leukemia (Tγδ LGLL) is a rare lymphoproliferative disease, scantily described in literature. A deep-analysis, in an initial cohort of 9 Tγδ LGLL compared to 23 healthy controls, shows that Tγδ LGLL dominant clonotypes are mainly public and exhibit different V-(D)-J γ/δ usage between patients with symptomatic and indolent Tγδ neoplasm. Moreover, some clonotypes share the same rearranged sequence. Data obtained in an enlarged cohort (n = 36) indicate the importance of a combined evaluation of immunophenotype and STAT mutational profile for the correct management of patients with Tγδ cell expansions. In fact, we observe an association between Vδ2/Vγ9 clonality and indolent course, while Vδ2/Vγ9 negativity correlates with symptomatic disease. Moreover, the 7 patients with STAT3 mutations have neutropenia and a CD56-/Vδ2- phenotype, and the 3 cases with STAT5B mutations display an asymptomatic clinical course and CD56/Vδ2 expression. All these data indicate that biological characterization is needed for Tγδ-cell neoplasm definition

CK1α/RUNX2 Axis in the Bone Marrow Microenvironment: A Novel Therapeutic Target in Multiple Myeloma

SIMPLE SUMMARY: Multiple myeloma (MM) is an incurable disease for which novel therapeutic approaches targeting the malignant cells and the associated bone disease are urgently needed. CK1α is a protein kinase that plays a crucial role in the signaling network that sustains plasma cell (PC) survival and bone disease. This protein regulates Wnt/β-catenin signaling, which is fundamental for both MM cell survival and mesenchymal stromal cell (MSC) osteogenic differentiation. In this study, we investigated its involvement in MM–MSC cross-talk. We found that, by lowering CK1α expression levels in co-cultures of MM and MSC cells, expression of RUNX2—the master regulator of osteogenic differentiation—was regulated differently in the two cell types. Our data suggest the possibility of using a specific CK1α inhibitor as part of a novel therapeutic approach to selectively kill malignant PCs and overcome the blocking of osteogenic differentiation induced by MM cells in MSCs. ABSTRACT: Multiple myeloma (MM) is a malignant plasma cell (PC) neoplasm, which also displays pathological bone involvement. Clonal expansion of MM cells in the bone marrow causes a perturbation of bone homeostasis that culminates in MM-associated bone disease (MMABD). We previously demonstrated that the S/T kinase CK1α sustains MM cell survival through the activation of AKT and β-catenin signaling. CK1α is a negative regulator of the Wnt/β-catenin cascade, the activation of which promotes osteogenesis by directly stimulating the expression of RUNX2, the master gene regulator of osteoblastogenesis. In this study, we investigated the role of CK1α in the osteoblastogenic potential of mesenchymal stromal cells (MSCs) and its involvement in MM–MSC cross-talk. We found that CK1α silencing in in vitro co-cultures of MMs and MSCs modulated RUNX2 expression differently in PCs and in MSCs, mainly through the regulation of Wnt/β-catenin signaling. Our findings suggest that the CK1α/RUNX2 axis could be a potential therapeutic target for constraining malignant PC expansion and supporting the osteoblastic transcriptional program of MSCs, with potential for ameliorating MMABD. Moreover, considering that Lenalidomide treatment leads to MM cell death through Ikaros, Aiolos and CK1α proteasomal degradation, we examined its effects on the osteoblastogenic potential of MSC compartments