Identification of <i>PSMB4</i> and <i>PSMD4</i> as novel target genes correlated with 1q21 amplification in patients with smoldering myeloma and multiple myeloma

Multiple myeloma (MM) is a malignant plasma cell (PC) dyscrasia characterized by heterogeneous biological features and genetic alterations, resulting in a wide range of disease courses.1,2 Despite all the therapeutic strategies developed in the last three decades, MM is still incurable, and almost all patients will inevitably experience disease progression and eventually relapse.3 Among all the genetic abnormalities, the amplification of the 1q21 region is one of the most frequent cytogenetic abnormalities occurring in malignant PC and it has become a new prognostic factor in MM patients.4,5 The incidence of gain and/or amplification of the 1q21 locus (1q21+) increases with disease progression. It can be detected in around 30-45% of patients with smoldering MM (SMM) and newly diagnosed MM (NDMM), and in around 70% of relapsed/refractory MM patients (RRMM).6 The impact of 1q21 on disease progression at an early stage has not been widely investigated. A few studies have suggested that the acquisition of extra 1q21 copies may play a role in disease progression.7,8 In fact, SMM patients with 1q21+ may be more likely to progress to MM than patients without 1q21+.8 Recent studies have demonstrated that the 1q21 copy number has a different impact on the responsiveness to MM treatments, especially proteasome inhibition (PI).9 PI is a well-established anti-cancer treatment approach used in MM. Throughout the years, the implementation of PI drugs as part of standard MM therapy has continued to improve the quality of life and clinical outcomes of MM patients. Furthermore, additional copies of 1q21 have been associated with PI resistance and recurrence of the disease in patients with 1q21+, limiting the long-term medical utility of PI.9,10 Recent studies have demonstrated that patients with 1q21+ treated with combination treatment with bortezomib (Bor) have inferior progression-free survival and overall survival compared to patients who do not present 1q21+.11 Similar results were observed when patients harboring 1q21 ampliSeveral genes are known to be deregulated upon the amplification of the 1q21 locus;9 nonetheless, the pathogenic and their possible role as druggable targets is not fully understood. In our study, we analyzed primary MM bone marrow (BM) PC from both SMM and NDMM patients to identify gene

Identification of clinical-biological features of newly diagnosed early relapse multiple myeloma patients eligible for autologous stem cell transplantation

: A portion of multiple myeloma (MM) patients relapse early or do not respond to first line treatment. Identification of possible clinical and or biological features of these patients remains an unmet medical need. In this study we assesed the predictive markers for early relapse MM, defined as a progressive disease that occurred within 18 months, from autologoust stem cell transplantation (ASCT) in MM patients who did not have primary refractory disease. 74 consecutive MM patients were included in the study that received intensive therapy with ASCT. The study was able to identify the main features of newly diagnosed ER MM patients eligible for ASCT identifying the IgA isotype and the R2-ISS score system as the main predictive prognostic factors for ER in this cohort of MM patients

Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells

<div><p>Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.</p></div

Analysis of vinculin, vimentin and cadherins gene expression by real time RT-PCR.

<p>The panels show the relative expression levels of the indicated transcripts in 46BR.lG1 (gray bars) and 7A3 cells (black bars) before (-) and after (+) incubation with 10 μM KU-55933. Gene transcripts have been internally normalized versus RPLP0 expression levels. Data are shown as mean ± SEM of four independent experiments. CDH: cadherin, VCL: vinculin, VIM: vimentin. * P < 0 .05, ** P < 0.01, *** P < 0.001.</p

Differential expression of cadherin 13 and cadherin 4 proteins in 46BR.1G1 and 31W cells.

<p>Cell lysates from 46BR.1G1 and 31W cells were analyzed by Western blotting with antibodies against the indicated proteins.</p

Enrichment analysis of IPA molecular function categories.

<p>Enrichment analysis of IPA molecular function categories.</p

CAD204520 Targets NOTCH1 PEST Domain Mutations in Lymphoproliferative Disorders

NOTCH1 PEST domain mutations are often seen in hematopoietic malignancies, including T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). These mutations play a key role in the development and progression of lymphoproliferative tumors by increasing the Notch signaling and, consequently, promoting cell proliferation, survival, migration, and suppressing apoptosis. There is currently no specific treatment available for cancers caused by NOTCH1 PEST domain mutations. However, several NOTCH1 inhibitors are in development. Among these, inhibition of the Sarco-endoplasmic Ca2+-ATPase (SERCA) showed a greater effect in NOTCH1-mutated tumors compared to the wild-type ones. One example is CAD204520, a benzimidazole derivative active in T-ALL cells harboring NOTCH1 mutations. In this study, we preclinically assessed the effect of CAD204520 in CLL and MCL models and showed that NOTCH1 PEST domain mutations sensitize cells to the anti-leukemic activity mediated by CAD204520. Additionally, we tested the potential of CAD204520 in combination with the current first-line treatment of CLL, venetoclax, and ibrutinib. CAD204520 enhanced the synergistic effect of this treatment regimen only in samples harboring the NOTCH1 PEST domain mutations, thus supporting a role for Notch inhibition in these tumors. In summary, our work provides strong support for the development of CAD204520 as a novel therapeutic approach also in chronic lymphoproliferative disorders carrying NOTCH1 PEST domain mutations, emerging as a promising molecule for combination treatment in this aggressive subset of patients

LigI-deficient 46BR.1G1 cells adhere more efficiently to the plate than complemented 7A3 cells.

<p>Cells were plated on 96-well plate and allowed to adhere for 30 minutes before fixing. Cells were stained with Crystal Violet, solubilized with acetic acid and quantified by measuring the OD at 620 nm. Data are shown as mean ± SEM of four independent experiments.</p

Differential expression of cadherin 13 and cadherin 4 proteins in 46BR.1G1 and 7A3 cells.

<p>(A) Cell lysates from 46BR.1G1 and 7A3 cells were analyzed by Western blotting with anti-cadherin 13, anti-cadherin 4, and anti-α-tubulin antibodies. (B) Quantification of the assay was performed by densitometric analysis with NIH ImageJ 1.43 program. Bars show mean ± SEM of three independent experiments.</p