RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening.

BACKGROUND: Personalized disease models are crucial for evaluating how diseased cells respond to treatments, especially in case of innovative biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells (nKPCs). METHODS: EVs were isolated from nKPCs derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients' urine and a line of Alport syndrome patient podocytes were characterized and used to assess albumin permeability in response to nKPC-EVs or various drugs. RNA sequencing was conducted to identify commonly modulated pathways after nKPC-EV treatment. siRNA transfection was used to demonstrate the involvement of SUMO1 and SENP2 in the modulation of permeability. RESULTS: Treatment with the nKPC-EVs significantly reduced permeability across all the steroid-resistant patients-derived and Alport syndrome-derived podocytes. At variance, podocytes appeared unresponsive to standard pharmacological treatments, with the exception of one line, in alignment with the patient's clinical response at 48 months. By RNA sequencing, only two genes were commonly upregulated in nKPC-EV-treated genetically altered podocytes: small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2). SUMO1 and SENP2 downregulation increased podocyte permeability confirming the role of the SUMOylation pathway. CONCLUSIONS: nKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocytes with genetic dysfunction, through modulation of SUMOylation, an important pathway for the stability of podocyte slit diaphragm proteins. Our findings also suggest the feasibility of developing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

Macrophage- and BCR-derived but not TLR-derived signals support the growth of CLL and Richter syndrome murine models in vivo.

A large amount of circumstantial evidence has accumulated suggesting that Toll-like receptor (TLR) signals are involved in driving chronic lymphocytic leukemia (CLL) cell proliferation, but direct in vivo evidence for this is still lacking. We have now further addressed this possibility by pharmacologically inhibiting or genetically inactivating the TLR pathway in murine CLL and human Richter syndrome (RS) patient-derived xenograft (PDX) cells. Surprisingly, we show that pharmacologic inhibition of TLR signaling by treatment with an IRAK1/4 inhibitor delays the growth of the transplanted malignant cells in recipient mice, but genetic inactivation of the same pathway by CRISPR/Cas9-mediated disruption of IRAK4 or its proximal adaptor MyD88 has no effect. We further show that treatment with the IRAK1/4 inhibitor results in depletion of macrophages and demonstrate that these cells can support the survival and enhance the proliferation of both murine Eµ-TCL1 leukemia and human RS cells. We also show that genetic disruption of the B-cell receptor (BCR) by CRISPR/Cas9 editing of the immunoglobulin M constant region gene inhibits the growth of human RS-PDX cells in vivo, consistent with our previous finding with murine Eµ-TCL1 leukemia cells. Finally, we show that genetic disruption of IRAK4 does not result in negative selection of human CLL cell lines xenografted in immunodeficient mice. The obtained data suggest that TLR signals are unlikely to represent a major driver of CLL/RS cell proliferation and provide further evidence that signals from macrophages and the BCR promote the growth and survival of CLL and RS cells in vivo.

The role of genetic testing in the diagnostic workflow of pediatric patients with kidney diseases: the experience of a single institution.

PURPOSE: Inherited kidney diseases are among the leading causes of kidney failure in children, resulting in increased mortality, high healthcare costs and need for organ transplantation. Next-generation sequencing technologies can help in the diagnosis of rare monogenic conditions, allowing for optimized medical management and therapeutic choices. METHODS: Clinical exome sequencing (CES) was performed on a cohort of 191 pediatric patients from a single institution, followed by Sanger sequencing to confirm identified variants and for family segregation studies. RESULTS: All patients had a clinical diagnosis of kidney disease: the main disease categories were glomerular diseases (32.5%), ciliopathies (20.4%), CAKUT (17.8%), nephrolithiasis (11.5%) and tubular disease (10.5%). 7.3% of patients presented with other conditions. A conclusive genetic test, based on CES and Sanger validation, was obtained in 37.1% of patients. The highest detection rate was obtained for ciliopathies (74.4%), followed by nephrolithiasis (45.5%), tubular diseases (45%), while most glomerular diseases and CAKUT remained undiagnosed. CONCLUSIONS: Results indicate that genetic testing consistently used in the diagnostic workflow of children with chronic kidney disease can (i) confirm clinical diagnosis, (ii) provide early diagnosis in the case of inherited conditions, (iii) find the genetic cause of previously unrecognized diseases and (iv) tailor transplantation programs.

Significant Long-Term Prevention of High Sensitization After Kidney Allograft Failure by Maintaining Calcineurin Inhibitor-Based Immunosuppression.

INTRODUCTION: Broad national or international programs contribute to mitigating the expected longer waiting list (WL) time for sensitized patients but with minor benefits for highly sensitized subjects. Therefore, strategies to prevent high sensitization are urgently required. In this study, we investigated the risk of developing highly sensitized patients with different immunosuppressive (IS) handling after kidney allograft failure (KAF). METHODS: Data from 185 patients with KAF, retransplanted/relisted from 2010 to 2020 in two regions of Italy that share the same regional WL, were analyzed. Patients were categorized according to IS management at 12 months after KAF as follows: patients maintaining IS with calcineurin inhibitors (CNI) (late withdrawal group [LWG], n = 58) and those who withdrew all IS therapy or were on steroids only (early withdrawal group [EWG], n = 127). RESULTS: Patients in the LWG showed lower panel reactive antibodies (PRA) at 12 (29.0% vs. 85.5%, p < 0.001) and 24 months (61.0% vs. 91.0%, p = 0.001), reduced risk of high sensitization (PRA ≥90%) at 12 (9.4% vs. 40.7%, p < 0.001, OR = 0.15) and 24 months (25.6% vs. 57.3%, p = 0.001, OR = 0.26) and almost no very high sensitization (PRA ≥ 98%) at 12 months (1.9% vs. 18.6%, p = 0.003, OR = 0.08) after KAF. In the LWG subgroup analysis, patients who maintained IS for up to 24 months after KAF did not show very high sensitization. The LWG showed shorter active WL times (406 vs. 813 days, p = 0.001) without an increased risk of complications. CONCLUSIONS: CNI maintenance for at least 12 months after KAF could be a useful approach to prevent high sensitization and reduce WL times in patients who are offered retransplantation, without a higher burden of complications.

Matching clinical and genetic data in pediatric patients at risk of developing cystic kidney disease.

BACKGROUND: Cystic kidney disease is a heterogeneous group of hereditary and non-hereditary pathologic conditions, associated with the development of renal cysts. These conditions may be present both in children and adults. Cysts can even be observed already during the prenatal age, and pediatric patients with cysts need to be clinically monitored. An early clinical and genetic diagnosis is therefore mandatory for optimal patient management. The aim of this study was to perform genetic analyses in patients with echographic evidence of kidney cysts to provide an early molecular diagnosis. METHODS: A cohort of 70 pediatric patients was enrolled and clinically studied at the time of first recruitment and at follow-up. Genetic testing by clinical exome sequencing was performed and a panel of genes responsible for "cystic kidneys" was analyzed to identify causative variants. Sanger validation and segregation studies were exploited for the final classification of the variants and accurate genetic counseling. RESULTS: Data showed that 53/70 of pediatric patients referred with a clinical suspicion of cystic kidney disease presented a causative genetic variant. In a significant proportion of the cohort (24/70), evidence of hyper-echogenic/cystic kidneys was already present in the prenatal period, even in the absence of a positive family history. CONCLUSIONS: This study suggests that cystic kidney disease may develop since the very early stages of life and that screening programs based on ultrasound scans and genetic testing play a critical role in diagnosis, allowing for better clinical management and tailored genetic counseling to the family.

Decreased trabecular bone score in patients affected by Fabry disease.

BACKGROUND: Fabry disease (FD) is an inherited X-linked lysosomal storage disease characterized by increased risk of osteoporosis and fractures. The impact of FD on clinical measures of bone quality is unknown. This considered, aim of our study was to evaluate whether trabecular bone microarchitecture, measured by trabecular bone score (TBS), is altered in patients with FD compared to control subjects. METHODS: This retrospective monocentric study enrolled 14 patients (M/F 1/1, median age 46 [37-63] years, range 31-72 years) newly diagnosed with FD between January 2016 and July 2023 who underwent dual-energy X-ray absorptiometry (DXA) image at the time of diagnosis and 42 matched controls. In all subjects, data about bone mineral density (BMD) and lumbar spine TBS were collected and total calcium, parathyroid hormone (PTH), 25(OH) vitamin D, alkaline phosphatase (ALP), creatinine and estimated glomerular filtration rate (eGFR) were evaluated. In subjects with FD, globotriaosylsphingosine (lyso-Gb3), 24-hour proteinuria and albumin-creatinine ratio were also assessed. RESULTS: Patients with FD presented significantly lower lumbar spine TBS (1.29 [1.22-1.38] vs. 1.42 [1.39-1.47], p < 0.001) and lower lumbar spine BMD (0.916 ± 0.166 vs. 1.031 ± 0.125 g/cm2, p = 0.008) compared to controls; moreover, FD was shown to be an independent risk factor for both low lumbar spine TBS (ß = -0.118, p < 0.001) and BMD (ß = -0.115, p = 0.009). No differences were found in serum calcium, ALP, 25(OH) vitamin D and eGFR in both groups, but FD patients had significantly higher PTH levels compared to controls (p = 0.016). Finally, 8 patients with FD presented either moderately or severely increased albuminuria and only 2 patients presented normal lyso-Gb3 levels. CONCLUSION: Patients affected by FD present significantly lower lumbar spine TBS and BMD compared to controls. Our findings strongly support the importance of carrying out a thorough evaluation of bone status in all patients affected by FD at baseline.

Genomic and Transcriptomic Profile of HNF1A-Mutated Liver Adenomas Highlights Molecular Signature and Potential Therapeutic Implications.

Hepatocellular adenomas (HAs) are tumors that can develop under different conditions, including in patients harboring a germline mutation in HNF1A. However, little is known about the pathogenesis of such disease. This work aims to better define what mechanisms lie under the development of this condition. Six HAs were sampled from the liver of a 17-year-old male affected by diabetes and multiple hepatic adenomatosis harboring the heterozygous pathogenic germline variant c.815G>A, p.(Arg272His) in HNF1A, which has a dominant negative effect. All HAs were molecularly characterized. Four of them were shown to harbor a second somatic HNF1A variant and one had a mutation in the ARID1A gene, while no additional somatic changes were found in the remaining HA and normal parenchyma. A transcriptomic profile of the same HA samples was also performed. HNF1A biallelic mutations were associated with the up-regulation of several pathways including the tricarboxylic acid cycle, the metabolism of fatty acids, and mTOR signaling while angiogenesis, endothelial and vascular proliferation, cell migration/adhesion, and immune response were down-regulated. Contrariwise, in the tumor harboring the ARID1A variant, angiogenesis was up-modulated while fatty acid metabolism was down-modulated. Histological analyses confirmed the molecular data. Independently of the second mutation, energetic processes and cholesterol metabolism were up-modulated, while the immune response was down-modulated. This work provides a complete molecular signature of HNF1A-associated HAs, analyzing the association between specific HNF1A variants and the development of HA while identifying potential new therapeutic targets for non-surgical treatment.

Molecular insights into the interaction between human nicotinamide phosphoribosyltransferase and Toll-like receptor 4.

The secreted form of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes a key reaction in intracellular NAD biosynthesis, acts as a damage-associated molecular pattern triggering Toll-like receptor 4 (TLR4)-mediated inflammatory responses. However, the precise mechanism of interaction is unclear. Using an integrated approach combining bioinformatics and functional and structural analyses, we investigated the interaction between NAMPT and TLR4 at the molecular level. Starting from previous evidence that the bacterial ortholog of NAMPT cannot elicit the inflammatory response, despite a high degree of structural conservation, two positively charged areas unique to the human enzyme (the α1-α2 and ß1-ß2 loops) were identified as likely candidates for TLR4 binding. However, alanine substitution of the positively charged residues within these loops did not affect either the oligomeric state or the catalytic efficiency of the enzyme. The kinetics of the binding of wildtype and mutated NAMPT to biosensor-tethered TLR4 was analyzed. We found that mutations in the α1-α2 loop strongly decreased the association rate, increasing the KD value from 18 nM, as determined for the wildtype, to 1.3 µM. In addition, mutations in the ß1-ß2 loop or its deletion increased the dissociation rate, yielding KD values of 0.63 and 0.22 µM, respectively. Mutations also impaired the ability of NAMPT to trigger the NF-κB inflammatory signaling pathway in human cultured macrophages. Finally, the involvement of the two loops in receptor binding was supported by NAMPT-TLR4 docking simulations. This study paves the way for future development of compounds that selectively target eNAMPT/TLR4 signaling in inflammatory disorders.

Synergistic efficacy of the dual PI3K-δ/γ inhibitor duvelisib with the Bcl-2 inhibitor venetoclax in Richter syndrome PDX models.

A small subset of cases of chronic lymphocytic leukemia undergoes transformation to diffuse large B-cell lymphoma, Richter syndrome (RS), which is associated with a poor prognosis. Conventional chemotherapy results in limited responses, underlining the need for novel therapeutic strategies. Here, we investigate the ex vivo and in vivo efficacy of the dual phosphatidylinositol 3-kinase-δ/γ (PI3K-δ/γ) inhibitor duvelisib (Duv) and the Bcl-2 inhibitor venetoclax (Ven) using 4 different RS patient-derived xenograft (PDX) models. Ex vivo exposure of RS cells to Duv, Ven, or their combination results in variable apoptotic responses, in line with the expression levels of target proteins. Although RS1316, IP867/17, and RS9737 cells express PI3K-δ, PI3K-γ, and Bcl-2 and respond to the drugs, RS1050 cells, expressing very low levels of PI3K-γ and lacking Bcl-2, are fully resistant. Moreover, the combination of these drugs is more effective than each agent alone. When tested in vivo, RS1316 and IP867/17 show the best tumor growth inhibition responses, with the Duv/Ven combination leading to complete remission at the end of treatment. The synergistic effect of Duv and Ven relies on the crosstalk between PI3K and apoptotic pathways occurring at the GSK3ß level. Indeed, inhibition of PI3K signaling by Duv results in GSK3ß activation, leading to ubiquitination and subsequent degradation of both c-Myc and Mcl-1, making RS cells more sensitive to Bcl-2 inhibition by Ven. This work provides, for the first time, a proof of concept of the efficacy of dual targeting of PI3K-δ/γ and Bcl-2 in RS and providing an opening for a Duv/Ven combination for these patients. Clinical studies in aggressive lymphomas, including RS, are under way. This trial was registered at www.clinicaltrials.gov as #NCT03892044.

B-cell receptor signaling and genetic lesions in TP53 and CDKN2A/CDKN2B cooperate in Richter transformation.

B-cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptors or T cells. Here, we report that BCR engagement of human and murine CLL cells induces several positive regulators of the cell cycle, but simultaneously induces the negative regulators CDKN1A, CDKN2A, and CDKN2B, which block cell-cycle progression. We further show that introduction of genetic lesions that downregulate these cell-cycle inhibitors, such as inactivating lesions in CDKN2A, CDKN2B, and the CDKN1A regulator TP53, leads to more aggressive disease in a murine in vivo CLL model and spontaneous proliferation in vitro that is BCR dependent but independent of costimulatory signals. Importantly, inactivating lesions in CDKN2A, CDKN2B, and TP53 frequently co-occur in Richter syndrome (RS), and BCR stimulation of human RS cells with such lesions is sufficient to induce proliferation. We also show that tumor cells with combined TP53 and CDKN2A/2B abnormalities remain sensitive to BCR-inhibitor treatment and are synergistically sensitive to the combination of a BCR and cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor both in vitro and in vivo. These data provide evidence that BCR signals are directly involved in driving CLL cell proliferation and reveal a novel mechanism of Richter transformation.

ROR1 targeting with the antibody-drug conjugate VLS-101 is effective in Richter syndrome patient-derived xenograft mouse models.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.

The immunomodulatory molecule TIGIT is expressed by chronic lymphocytic leukemia cells and contributes to anergy.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory checkpoint receptor that negatively regulates Tcell responses. CD226 competes with TIGIT for binding to the CD155 ligand, delivering a positive signal to the T cell. Here we studied the expression of TIGIT and CD226 in a cohort of 115 patients with chronic lymphocytic leukemia (CLL) and report expression of TIGIT and CD226 by leukemic cells. By devising a TIGIT/CD226 ratio, we showed that CLL cells favoring TIGIT over CD226 are typical of a more indolent disease, while those favoring CD226 are characterized by a shorter time to first treatment and shorter progression-free survival after first treatment. TIGIT expression was inversely correlated to the B-cell receptor (BCR) signaling capacity, as determined by studying BTK phosphorylation, cell proliferation and interleukin- 10 production. In CLL cells treated with ibrutinib, in which surface IgM and BCR signaling capacity are temporarily increased, TIGIT expression was downmodulated, in line with data indicating transient recovery from anergy. Lastly, cells from patients with Richter syndrome were characterized by high levels of CD226, with low to undetectable TIGIT, in keeping with their high proliferative drive. Together, these data suggest that TIGIT contributes to CLL anergy by downregulating BCR signaling, identifying novel and actionable molecular circuits regulating anergy and modulating CLL cell functions.

Rejection markers in kidney transplantation: do new technologies help children?

Recent insights in allorecognition and graft rejection mechanisms revealed a more complex picture than originally considered, involving multiple pathways of both adaptive and innate immune response, supplied by efficient inflammatory synergies. Current pillars of transplant monitoring are serum creatinine, proteinuria, and drug blood levels, which are considered as traditional markers, due to consolidated experience, low cost, and widespread availability. The most diffuse immunological biomarkers are donor-specific antibodies, which are included in routine post-transplant monitoring in many centers, although with some reproducibility issues and interpretation difficulties. Confirmed abnormalities in these traditional biomarkers raise the suspicion for rejection and guide the indication for graft biopsy, which is still considered the gold standard for rejection monitoring. Rapidly evolving new "omic" technologies have led to the identification of several novel biomarkers, which may change the landscape of transplant monitoring should their potential be confirmed. Among them, urinary chemokines and measurement of cell-free DNA of donor origin are perhaps the most promising. However, at the moment, these approaches remain highly expensive and cost-prohibitive in most settings, with limited clinical applicability; approachable costs upon technology investments would speed their integration. In addition, transcriptomics, metabolomics, proteomics, and the study of blood and urinary extracellular vesicles have the potential for early identification of subclinical rejection with high sensitivity and specificity, good reproducibility, and for gaining predictive value in an affordable cost setting. In the near future, information derived from these new biomarkers is expected to integrate traditional tools in routine use, allowing identification of rejection prior to clinical manifestations and timely therapeutic intervention. This review will discuss traditional, novel, and invasive and non-invasive biomarkers, underlining their strengths, limitations, and present or future applications in children.

Extracellular Vesicles in Liver Transplantation: Current Evidence and Future Challenges.

Extracellular vesicles (EVs) are emerging as a promising field of research in liver disease. EVs are small, membrane-bound vesicles that contain various bioactive molecules, such as proteins, lipids, and nucleic acids and are involved in intercellular communication. They have been implicated in numerous physiological and pathological processes, including immune modulation and tissue repair, which make their use appealing in liver transplantation (LT). This review summarizes the current state of knowledge regarding the role of EVs in LT, including their potential use as biomarkers and therapeutic agents and their role in graft rejection. By providing a comprehensive insight into this emerging topic, this research lays the groundwork for the potential application of EVs in LT.

Targeting metabolic reprogramming in metastatic melanoma: The key role of nicotinamide phosphoribosyltransferase (NAMPT).

Cancer cells rewire their metabolism to support proliferation, growth and survival. In metastatic melanoma the BRAF oncogenic pathway is a master regulator of this process, highlighting the importance of metabolic reprogramming in the pathogenesis of this tumor and offering potential therapeutic approaches. Metabolic adaptation of melanoma cells generally requires increased amounts of NAD+, an essential redox cofactor in cellular metabolism and a signaling molecule. Nicotinamide phosphoribosyltransferase (NAMPT) is the most important NAD+ biosynthetic enzyme in mammalian cells and a direct target of the BRAF oncogenic signaling pathway. These findings suggest that NAMPT is an attractive new therapeutic target, particularly in combination strategies with BRAF or MEK inhibitors. Here we review current knowledge on how oncogenic signaling reprograms metabolism in BRAF-mutated melanoma, and discuss how NAMPT/NAD+ axis contributes to these processes. Lastly, we present evidence supporting a role of NAMPT as a novel therapeutic target in metastatic melanoma.

Donor-derived acute myeloid leukemia in solid organ transplantation.

We report the transmission of acute myeloid leukemia (AML) undetected at donation from a deceased organ donor to two kidneys and one liver recipients. We reviewed the medical records, and performed molecular analyses and whole exome sequencing (WES) to ascertain AML donor origin and its molecular evolution. The liver recipient was diagnosed 11 months after transplantation and died from complications 2 months later. The two kidney recipients (R1 and R2) were diagnosed 19 and 20 months after transplantation and both received treatment for leukemia. R1 died of complications 11 months after diagnosis, while R2 went into complete remission for 44 months, before relapsing. R2 died 10 months later of complications from allogenic bone marrow transplantation. Microsatellite analysis demonstrated donor chimerism in circulating cells from both kidney recipients. Targeted molecular analyses and medical records revealed NPM1 mutation present in the donor and recipients, while FLT3 was mutated only in R1. These findings were confirmed by WES, which revealed additional founder and clonal mutations, and HLA genomic loss in R2. In conclusion, we report the first in-depth genomic analysis of AML transmission following solid organ transplantation, revealing distinct clonal evolution, and providing a potential molecular explanation for tumor escape.

Genomic Mismatch at LIMS1 Locus and Kidney Allograft Rejection.

BACKGROUND: In the context of kidney transplantation, genomic incompatibilities between donor and recipient may lead to allosensitization against new antigens. We hypothesized that recessive inheritance of gene-disrupting variants may represent a risk factor for allograft rejection. METHODS: We performed a two-stage genetic association study of kidney allograft rejection. In the first stage, we performed a recessive association screen of 50 common gene-intersecting deletion polymorphisms in a cohort of kidney transplant recipients. In the second stage, we replicated our findings in three independent cohorts of donor-recipient pairs. We defined genomic collision as a specific donor-recipient genotype combination in which a recipient who was homozygous for a gene-intersecting deletion received a transplant from a nonhomozygous donor. Identification of alloantibodies was performed with the use of protein arrays, enzyme-linked immunosorbent assays, and Western blot analyses. RESULTS: In the discovery cohort, which included 705 recipients, we found a significant association with allograft rejection at the LIMS1 locus represented by rs893403 (hazard ratio with the risk genotype vs. nonrisk genotypes, 1.84; 95% confidence interval [CI], 1.35 to 2.50; P = 9.8×10-5). This effect was replicated under the genomic-collision model in three independent cohorts involving a total of 2004 donor-recipient pairs (hazard ratio, 1.55; 95% CI, 1.25 to 1.93; P = 6.5×10-5). In the combined analysis (discovery cohort plus replication cohorts), the risk genotype was associated with a higher risk of rejection than the nonrisk genotype (hazard ratio, 1.63; 95% CI, 1.37 to 1.95; P = 4.7×10-8). We identified a specific antibody response against LIMS1, a kidney-expressed protein encoded within the collision locus. The response involved predominantly IgG2 and IgG3 antibody subclasses. CONCLUSIONS: We found that the LIMS1 locus appeared to encode a minor histocompatibility antigen. Genomic collision at this locus was associated with rejection of the kidney allograft and with production of anti-LIMS1 IgG2 and IgG3. (Funded by the Columbia University Transplant Center and others.).

Tumors carrying BRAF-mutations over-express NAMPT that is genetically amplified and possesses oncogenic properties.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways. METHODS: Exploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose-response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself. RESULTS: The first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT. CONCLUSIONS: Overall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.

Flow cytometric evaluation of measurable residual disease in chronic lymphocytic leukemia: Where do we stand?

Measurable residual disease (MRD) has emerged as a relevant parameter of response to therapy in chronic lymphocytic leukemia (CLL). Although several methods have been developed, flow cytometry has emerged as the most useful and standardized approach to measure and quantify MRD. The improved sensitivity of MRD measurements has been paralleled by the development of more effective therapeutic strategies for CLL, increasing the applicability of MRD detection in this setting. Chemotherapy and chemoimmunotherapy have firstly demonstrated their ability to obtain a deep MRD. Combined targeted therapies are also demonstrating a high molecular response rate and prospective trials are exploring the role of MRD to guide the duration of treatment in this setting. In this review we briefly summarize what we have learned about MRD with emphasis on its flow cytometric detection.