CD38-Induced Metabolic Dysfunction Primes Multiple Myeloma Cells for NAD+-Lowering Agents.

Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

Apoptosis reprogramming triggered by splicing inhibitors sensitizes multiple myeloma cells to Venetoclax treatment.

Identification of novel vulnerabilities in the context of therapeutic resistance is emerging as a key challenge for cancer treatment. Recent studies have detected pervasive aberrant splicing in cancer cells, supporting its targeting for novel therapeutic strategies. Here, we evaluated the expression of several spliceosome machinery components in multiple myeloma (MM) cells and the impact of splicing modulation on tumor cell growth and viability. A comprehensive gene expression analysis confirmed the reported deregulation of spliceosome machinery components in MM cells, compared to normal plasma cells from healthy donors, with its pharmacological and genetic modulation resulting in impaired growth and survival of MM cell lines and patient-derived malignant plasma cells. Consistent with this, transcriptomic analysis revealed deregulation of BCL2 family members, including decrease of anti-apoptotic long form of myeloid cell leukemia-1 (MCL1) expression, as crucial for "priming" MM cells for Venetoclax activity in vitro and in vivo, irrespective of t(11;14) status. Overall, our data provide a rationale for supporting the clinical use of splicing modulators as a strategy to reprogram apoptotic dependencies and make all MM patients more vulnerable to BCL2 inhibitors.

RB1 Germline Variant Predisposing to a Rare Ovarian Germ Cell Tumor: A Case Report.

Malignant ovarian germ cell tumors (MOGCTs) are neoplasms of the ovary, of which, due to their rarity and heterogeneity, few is reported about genetic background and development. Here, we report a 18-years old patient diagnosed with an ovarian mixed germ cell tumor, without any previous history of malignancies, who has been treated with surgery and chemotherapy and died 4 years later due to peritoneal metastasis complications. Patient's blood DNA was screened for a panel of 52 cancer-related genes in order to identify predisposing aberrations to this rare cancer. The analysis discovered the uncharacterized c.2393G>A variant in RB1, the retinoblastoma gene, leading both to a missense change and a splicing perturbation of the RB1 transcript. The variant was found to be hypomorphic, damaging the C-terminal domain with a partially impaired protein function. The variant is inherited from the unaffected mother. Due to an imprinting mechanism, the maternal allele is ~3-fold more expressed than the paternal one. The parent-of-origin effect combined with the hypomorphic impact of the variant determines a rescue of sufficient tumor-suppressor activity to prevent retinoblastoma development but can predispose to other cancers in the adult age. In order to understand the somatic events acting on the germline predisposition we used the NGS-liquid biopsy covering 77 cancer driver genes. Using this approach, we detected deleterious mutations in TP53, SMAD4, FGFR3, and MSH2, indicative of a dis-regulation of cell cycle and DNA repair mechanisms pathways. In conclusion, we have pinpointed for the first time that an RB1 leaky variant, not leading to retinoblastoma because of its maternal origin, can predispose in adults to a very rare form of ovarian cancer and that the somatic disruption of few genes contributes to the tumor progression and aggressiveness.

Two-point-NGS analysis of cancer genes in cell-free DNA of metastatic cancer patients.

BACKGROUND: Although the efficacy of molecularly target agents in vitro, their use in routine setting is limited mainly to the use of anti-HER2 and antiEGFR agents in vivo. Moreover, core biopsy of a single cancer site may not be representative of the whole expanding clones and cancer molecular profile at relapse may differ with respect to the primary tumor. METHODS: We assessed the status of a large panel of cancer driver genes by cell-free DNA (cfDNA) analysis in a cohort of 68 patients with 13 different solid tumors at disease progression. Whenever possible, a second cfDNA analysis was performed after a mean of 2.5 months, in order to confirm the identified clone(s) and to check the correlation with clinical evolution. RESULTS: The approach was able to identify clones plausibly involved in the disease progression mechanism in about 65% of cases. A mean of 1.4 mutated genes (range 1-3) for each tumor was found. Point mutations in TP53, PIK3CA, and KRAS and copy number variations in FGFR3 were the gene alterations more commonly observed, with a rate of 48%, 20%, 16%, and 20%, respectively. Two-points-Next-Generation Sequencing (NGS) analysis demonstrated statistically significant correlation between allele frequency variation and clinical outcome (P = .026). CONCLUSIONS: Irrespective of the primary tumor mutational burden, few mutated genes are present at disease progression. Clinical outcome is consistent with variation of allele frequency of specific clones indicating that cfDNA two-point-NGS analysis of cancer driver genes could be an efficacy tool for precision oncology.

Usefulness and Limitations of Comprehensive Characterization of mRNA Splicing Profiles in the Definition of the Clinical Relevance of BRCA1/2 Variants of Uncertain Significance.

Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.

Evidence of predisposing epimutation in retinoblastoma.

Retinoblastoma (RB), which represents the most common childhood eye cancer, is caused by biallelic inactivation of RB1 gene. Promoter hypermethylation is quite frequent in RB tissues but conclusive evidence of soma-wide predisposing epimutations is currently scant. Here, 50 patients who tested negative for RB1 germline sequence alterations were screened for aberrant promoter methylation using methylation-specific MLPA. The assay, performed on blood, identified a sporadic patient with methylation of CpG106, absent in parents' DNA. Bisulfite pyrosequencing accurately quantified CpG methylation in blood DNA (mean ∼49%) and also confirmed the aberration in DNA isolated from oral mucosa although at lower levels (mean ∼34%). Using a tag-SNP, methylation was demonstrated to affect the maternal allele. Real-time qPCR demonstrated RB1 transcriptional silencing. In conclusion, we documented that promoter methylation can act as the first "hit" in Knudson's model. This mosaic epimutation mimics the effect of an inactivating mutation and phenocopies RB onset.

Parent-of-origin effect of hypomorphic pathogenic variants and somatic mosaicism impact on phenotypic expression of retinoblastoma.

Retinoblastoma is the most common eye cancer in children. Numerous families have been described displaying reduced penetrance and expressivity. An extensive molecular characterization of seven families led us to characterize the two main mechanisms impacting on phenotypic expression, as follows: (i) mosaicism of amorphic pathogenic variants; and (ii) parent-of-origin-effect of hypomorphic pathogenic variants. Somatic mosaicism for RB1 splicing variants (c.1960+5G>C and c.2106+2T>C), leading to a complete loss of function was demonstrated by high-depth NGS in two families. In both cases, the healthy carrier parent (one with retinoma) showed a variant frequency lower than that expected for a heterozygous individual, indicating a 56-60% mosaicism level. Previous evidences of a ~3-fold excess of RB1 maternal canonical transcript led us to hypothesize that this differential allelic expression could influence phenotypic outcome in families at risk for RB onset. Accordingly, in five families, we identified a higher tumor risk associated with paternally inherited hypomorphic pathogenic variants, namely a deletion resulting in the loss of 37 amino acids at the N-terminus (c.608-16_608del), an exonic substitution with a "leaky" splicing effect (c.1331A>G), a partially deleterious substitution (c.1981C>T) and a truncating C-terminal variant (c.2663+2T>C). The identification of these mechanisms changes the genetic/prenatal counseling and the clinical management of families, indicating a higher recurrence risk when the hypomorphic pathogenic variant is inherited from the father, and suggesting the need for second tumor surveillance in unaffected carriers at risk of developing adult-onset cancer such as osteosarcoma or leiomyosarcoma.