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Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Supplementary file contains a PDF version of supplementary figures S1-S7 and associated figure legends.

Supplementary file contains all supplementary tables S1-S16 in Excel format in the order in which they appear in the text.

This file contains supplemental additional experimental procedures, relevant references and 7 supplementary figures, including: S1 "Expression of EBV in RS samples"; S2 "Schematic representation of the RS-PDX models"; S3 "Disease engraftment and distribution of RS1316 i.v. model"; S4 "Phenotypic analysis of primary and derived-RS-PDXs"; S5 "Schematic diagram of proteins and corresponding mutations"; S6 "Differentially expressed genes in primary and RS-PDX"; S7 "Schematic sketch of the main mutated genes in our RS models and core signaling pathways they belong to and play a critical role". Supplementary tables contain additional information on sequencing results performed on primary and PDX-RS models.

Richter syndrome represents the evolution of chronic lymphocytic leukemia into an aggressive tumor, most commonly diffuse large B-cell lymphoma. The lack of in vitro and in vivo models has severely hampered drug testing in a disease that is poorly responsive to common chemoimmunotherapeutic combinations as well as to novel kinase inhibitors. Here we report for the first time the establishment and genomic characterization of two patient-derived tumor xenograft (PDX) models of Richter syndrome, RS9737 and RS1316. Richter syndrome xenografts were genetically, morphologically, and phenotypically stable and similar to the corresponding primary tumor. RS9737 was characterized by biallelic inactivation of CDKN2A and TP53, monoallelic deletion of 11q23 (ATM), and mutations of BTK, KRAS, EGR2, and NOTCH1. RS1316 carried trisomy 12 and showed mutations in BTK, KRAS, MED12, and NOTCH2. RNA sequencing confirmed that in both cases >80% of the transcriptome was shared between primary tumor and PDX. In line with the mutational profile, pathway analyses revealed overactivation of the B-cell receptor, NFκB, and NOTCH pathways in both tumors, potentially providing novel tumor targets. In conclusion, these two novel models of Richter syndrome represent useful tools to study biology and response to therapies of this highly aggressive and still incurable tumor.Significance: Two patient-derived xenograft models of Richter syndrome represent the first ex vivo model to study biology of the disease and to test novel therapeutic strategies. Cancer Res; 78(13); 3413–20. ©2018 AACR.

Supplementary Table 1, Figures 1-4 from Nicotinamide Blocks Proliferation and Induces Apoptosis of Chronic Lymphocytic Leukemia Cells through Activation of the p53/miR-34a/SIRT1 Tumor Suppressor Network

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.

BACKGROUND Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

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.

BACKGROUND: SARS-CoV-2 infection is heterogeneous in clinical presentation and disease evolution. To investigate whether immune response to the virus can be influenced by genetic factors, we compared HLA and AB0 frequencies in organ transplant recipients and waitlisted patients according to presence or absence of SARS-CoV-2 infection. METHODS: A retrospective analysis was performed on an Italian cohort composed by transplanted and waitlisted patients in a January 2002-March 2020 time frame. Data from this cohort were merged with the Italian registry of COVID subjects, evaluating infection status of transplanted and waitlisted patients. A total of 56304 cases were studied with the aim of comparing HLA and AB0 frequencies according to the presence (n=265, COVID) or absence (n=56 039, COVID) of SARS-CoV-2 infection. RESULTS: The cumulative incidence rate of COVID-19 was 0.112% in the Italian population and 0.462% in waitlisted/transplanted patients (OR=4.2, 95%CI [3.7-4.7], P

Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

Background Methylmalonic aciduria and homocystinuria, CblC type (OMIM #277400) is the most common disorder of cobalamin intracellular metabolism, an autosomal recessive disease, whose biochemical hallmarks are hyperhomocysteinemia, methylmalonic aciduria and low plasma methionine. Despite being a well-recognized disease for pediatricians, there is scarce awareness of its adult presentation. A thorough analysis and discussion of cobalamin C defect presentation in adult patients has never been extensively performed. This article reviews the published data and adds a new case of the latest onset of symptoms ever described for the disease. Results We present the emblematic case of a 45-year-old male, describing the diagnostic odyssey he ventured through to get to the appropriate treatment and molecular diagnosis. Furthermore, available clinical, biochemical and molecular data from 22 reports on cases and case series were collected, resulting in 45 adult-onset CblC cases, including our own. We describe the onset of the disease in adulthood, encompassing neurological, psychiatric, renal, ophthalmic and thromboembolic symptoms. In all cases treatment with intramuscular hydroxycobalamin was effective in reversing symptoms. From a molecular point of view adult patients are usually compound heterozygous carriers of a truncating and a non-truncating variant in the MMACHC gene. Conclusion Adult onset CblC disease is a rare disorder whose diagnosis can be delayed due to poor awareness regarding its presenting insidious symptoms and biochemical hallmarks. To avoid misdiagnosis, we suggest that adult onset CblC deficiency is acknowledged as a separate entity from pediatric late onset cases, and that the disease is considered in the differential diagnosis in adult patients with atypical hemolytic uremic syndromes and/or slow unexplained decline in renal function and/or idiopathic neuropathies, spinal cord degenerations, ataxias and/or recurrent thrombosis and/or visual field defects, maculopathy and optic disc atrophy. Plasma homocysteine measurement should be the first line for differential diagnosis when the disease is suspected. To further aid diagnosis, it is important that genes belonging to the intracellular cobalamin pathway are included within gene panels routinely tested for atypical hemolytic uremic syndrome and chronic kidney disorders.

Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naive B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.

Aim: Nicotinamide adenine dinucleotide (NAD+) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD+ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD+ levels in live cells and to address the issue of the compartmentalization of NAD+ biosynthesis, we exploited a recently described genetically encoded NAD+ biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM). Results: FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD+-producing enzyme in MM cells, was used to monitor NAD+ depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD+ precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD+ biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Innovation and Conclusions: Throughout this work, we validated the use of genetically encoded NAD+ biosensors to characterize subcellular distribution of NAD+ production routes in MM. The chance of real-time monitoring of NAD+ fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD+ pool manipulation in cancer and metabolic diseases.

Background and Aims 3MC syndrome is an autosomal recessive disorder encompassing a variable spectrum of abnormalities, among which facial dysmorphisms are characteristic. Mutations in genes which encode proteins involved in the lectin complement pathway MASP1, COLEC11 and recently COLEC10 have been identified in patients with 3MC syndrome, supporting their key role during human development. We present a 5 years old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, horseshoe kidneys, diastasis recti, umbilical depression and sacral dimple. The diagnosis was confirmed by sequencing of COLEC10 gene and the putative pathogenic variant was functionally validated through in vitro assays. Method COLEC10 gene was analyzed through Sanger sequencing. The secreted protein CL-L1 was investigated in the plasma of the patient and her parents by Western blot. The variant was introduced by a site-specific mutagenesis approach into a plasmid encoding wild-type human CL-L1. HeLa cells were then transfected with the mutated or wild-type plasmid and culture supernatant evaluated in a migration assay. Results A homozygous frameshift variant c.807_810delCTGT p.(Cys270Serfs*33) was identified in the patient. Segregation studies confirmed the parents’ carrier status for the variant. Functionally, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells are less sensitive to the mutant protein compared to the WT one, resulting in a reduced migratory response. Conclusion We report a patient affected by 3MC syndrome who, besides typical phenotypic signs, presents a patent ductus arteriosus, never described in association to COLEC10 before. The variant causative role was functionally confirmed in an in vitro assay, where the mutated protein failed to act as a chemo-attractant. We thus provide further evidence for CL-L1 role during embryonic development

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 GSK3β level. Indeed, inhibition of PI3K signaling by Duv results in GSK3β 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 (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.

Background and Aims next-generation sequencing (NGS) technologies are becoming a powerful diagnostic tool in precision medicine. Specifically, exome sequencing can help in the diagnosis of selected diseases, in their medical management and therapeutic choices. Inherited kidney diseases (IKD) are among the major causes for kidney failure, both in children and adults, resulting in increased mortality, high health care costs and need for organ transplantation. In addition, it is worth mentioning that a significant proportion of patients in the kidney transplant lacks a clear diagnosis. This subset of diseases may thus benefit from the application of NGS technology, as the simultaneous investigation of hundreds of genes can lead to the identification of causative variants in a vast population of patients. The aim of this study is to validate the use of a clinical exome sequencing approach in the diagnostic flow for kidney diseases leading to organ failure to i) confirm the clinical diagnosis, ii) find the genetic cause of previously unrecognized diseases and iii) improve the outcome of organ transplantation by excluding live-donors carrying the same mutational burden. Method 160 patients were recruited, directly or following a genetic counseling, exploiting a network of 21 nephrology centers spread across the Piedmont region, coordinated by the “Centro Regionale Trapianti (CRT)” of Torino. Patients were then evaluated for NGS eligibility. DNA extracted from blood samples was checked for integrity, quantified and used for library preparation. A clinical exome sequencing (CES) kit by Illumina was used, allowing for targeted capture, enrichment and sequencing of 6700 clinically relevant genes. Reads were aligned to hg37 reference genome using the Isaac enrichment tool and variants filtered using an ad-hoc set up pipeline of analysis. Results clinical exome sequencing was performed on a diagnostic cohort of 138 patients, both children (37.7%) and adults (62.3%), with a prevalence of male subjects (56.5%). The majority of the cohort (51.5%) presented a positive family history for kidney disease, while 22 patients were excluded from the study as organ failure was most likely the result of secondary events. The cohort was highly heterogeneous with 21% of patients presenting with ciliopathies, 18.1% with glomerular disease, 7.2% with tubular disease while the remaining cohort presented other diseases or was undiagnosed (44.3%). An ad hoc analytical pipeline was designed, based on selected genotype-phenotype correlation database, filter-in metrics, inheritance model and annotation of variants based on public databases and in-silico prediction tools. By adopting well defined criteria of recruitment and analysis, causative genes were identified in 61.6% of cases and in the 57.3% of cases results were in line with the original diagnostic hypothesis. Moreover, 50.8% of cases with organ failure for unknown reasons were solved with the identification of causative genes. Out of the 133 total variants found in the cohort, 63 were classified as pathogenic or likely pathogenic. The remaining 70 identified variants were annotated as variant of unknown significance and will be further investigated. Conclusion Taken together, these results show that CES is a powerful non-invasive tool for the genetic diagnosis of IKD. Identification of disease causative variants may represent a critical step for the diagnosis, clinical management of the patients, and potentially for optimal live-donor selection.

Background and Aims Autosomal dominant PKD determines formation of multiple cysts predominantly in the kidneys and usually becomes symptomatic during adulthood and can lead to renal failure. In contrast, in autosomal recessive PKD cysts occur in both the kidneys and the liver and usually presents an earlier onset. Obtaining genetic diagnosis is important to confirm clinical diagnosis and is required before treating with vasopressin 2 receptor blockers, which are the only drugs known to slow down the disease. Furthermore, in the case of kidney transplant from a living family member it is essential to exclude the presence of the mutation in the donor. We used clinical exome sequencing to provide genetic diagnosis to a cohort of patients with a clinical suspicion of PKD. Method 175 patients were referred to the Immunogenetics and Transplant Biology Service of the Turin University Hospital through a network of nephrology centers operating in the Piedmont region. Some patients were referred following genetic counseling. All patients signed an informed consent and the referring physicians provided relevant clinical data. DNA from eligible patients was extracted, checked for integrity, quantified and used for library preparation. A clinical exome sequencing (CES) kit by Illumina was used, allowing the analysis of 6,700 clinically relevant genes. Results Out of the 175 recruited patients eligible for CES, 38 (21.7%) had a clinical suspicion or diagnosis of PKD, with 50% of them presenting family history. The majority of the cohort was represented by male subjects (60.5%) and included both children (34.2%) and adults. The analytical approach was based on initial analysis of genes responsible for PKD (PKD1, PKD2 and PKHD1). If no mutation could be identified, analysis was then extended to a panel of 99 genes responsible for ciliopathies. This approach led to the identification of causative variants in 33/38 (86.8%) of the PKD cohort, while no variant could be identified in 5/38 patients. In 5/33 (15.2%) patients, mutations were inconclusive as found in heterozygosity in genes known to have an autosomal recessive mode of inheritance, while 27/33 (81.8%) were in line with the initial clinical suspicion/diagnosis. Of these, the majority was represented by missense mutations (12), followed by frameshift and nonsense mutations (6 each) and 3 splicing variants. As expected, the majority of mutations were found in PKD1 17/27 (63%), PKD2 3/27 (11.1%) and PKHD1 2/27 (7.4%). In these two latter patients, variants were found as compound heterozygosity. We also found mutations in other genes known to cause cysts, including TSC2 and CPT2. Of note, in 7 patients carrying PKD1 mutations, we found a second variant in PKD1 or PKHD1. Interestingly, when looking at patients characterized by kidney failure but lacking a clinical suspicion at recruitment or diagnosed with other phenotypes (66/175), we found variants in PKD1 and in PKD2 in 11 patients (9 and 2, respectively). Of all identified variants in PKD1, PKD2 and PKHD1 genes, 17.6% were annotated as pathogenic (C5), 41.2% were likely pathogenic (C4) and 41.2% were variants of unknown significance (C3). 19 variants in these genes were not previously reported. All the variants found in genes responsible for PKD were validated and confirmed by Sanger sequencing. Family segregation studies are ongoing. Finally, it is worth mentioning that in a portion of cases (5/38) with clinical and phenotypic features of PKD, supported also by a positive family history, we could not detect mutations in causative genes. These results may be explained by the presence of intronic variants, in line with data reported in literature. Conclusion These results demonstrate that CES may be applied to PKD patients to identify causative variants during their routine diagnostic flow. Furthermore, CES may be a useful tool to detect mutations in PKD-related genes in patients with undiagnosed diseases, considering its rapidly decreasing costs.

3MC syndrome is an autosomal recessive disorder encompassing four rare disorders previously known as the Malpuech, Michels, Mingarelli and Carnevale syndromes. They are characterized by a variable spectrum of abnormalities, including facial dysmorphisms, along with genital, limb and vesico-renal anomalies. The syndrome was originally attributed to mutations in MASP1 and COLEC11, which code for proteins involved in the lectin complement pathway. More recently, mutations in COLEC10, a third gene coding for collectin CL-L1, were identified in a limited number of patients with 3MC syndrome. Here we describe a 4-years-old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, sacral dimple and horseshoe kidney. Initial genetic analysis was based on clinical exome sequencing, where only MASP1 and COLEC11 genes are present, without evidence of pathogenic variants. Sanger sequencing of COLEC10 identified the homozygous frameshift variant c.807_810delCTGT; p.Cys270Serfs*33, which results in the loss of the natural stop codon. The resulting protein is 24 amino acids longer and lacks a conserved cysteine residue (Cys270), which could affect protein folding. Segregation studies confirmed that both parents were carriers for the variant: interestingly they originate from the same area of Apulia in southern Italy. Plasma levels of CL-L1 in the patient and her parents were within normal range, suggesting that this variant does not modify transcription or secretion. However, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells migrate significantly less in response to the mutant protein compared to the wild-type one.

The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL. Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy. We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050). These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels. αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used). Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells. Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®. ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany References Vaisitti T et al. Blood. 2021;137(24):3365-3377. Iannello A, et al. Blood. 2021;137(24):3378-3389. Disclosures Orlik: Heidelberg Pharma: Current Employment. Kulke: Heidelberg Pharma: Current Employment. Pahl: Heidelberg Pharma: Current Employment. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

BACKGROUND. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T, NK and NKT cells, sharing structural and mechanistic similarities with PD-1 and CTLA-4. TIGIT competes with CD226, its partner receptor, for the binding to CD155 ligand: signaling triggered upon CD155 binding to CD226 potentiates T cell receptor (TCR) signaling and CD8 + T cell cytotoxicity against tumor cells (positive signaling). On the contrary, concomitant TIGIT expression on the cell surface prevents CD226 activation either by sequestering CD155 or by impeding CD226 homodimerization and phosphorylation (negative signaling). Recently, TIGIT was shown to be expressed on the surface of normal memory B cells, where it could directly act to suppress T cell responses. No data are available on TIGIT or CD226 expression by chronic lymphocytic leukemia (CLL) cells. AIM AND METHODS. Our aim was to investigate expression of the TIGIT and CD226 receptors and of the CD155 ligand in a cohort of clinically and molecularly annotated CLL patient samples. To this end, we designed a multiparametric panel of antibodies for flow cytometry and examined expression of the TIGIT/CD226/CD155 axis in peripheral blood mononuclear cells (PBMC) from our patient cohort. To investigate the impact of TIGIT/CD226 engagement on B cell responses, purified leukemic B cells were activated either through the B cell receptor (BCR) using an αIgM polyclonal antibody or with CpG oligonucleotide and interleukin 15 (IL-15) to induce proliferation. In selected experiments, we added recombinant human (Rh) TIGIT-Fc or CD155-Fc chimeras and αTIGIT or αCD226 blocking antibodies to interfere with this axis. RESULTS. Surface expression of TIGIT, CD226 and CD155 was evaluated in a cohort of 115 CLL samples and compared to age- and sex-matched healthy subjects. Both TIGIT and CD226 were upregulated on leukemic B cells compared to normal B lymphocytes, while CD155 was expressed at lower levels. A similar trend was observed on CD4 + and CD8 + T lymphocytes. High-risk CLLs (unmutated IgV genes, unfavorable cytogenetics and advanced stage) were predominantly TIGIT low and CD226 high, indicating an unbalance towards "positive signaling". Results were confirmed by confocal microscopy analyses on lymph node (LN) biopsies, which showed i) an overall higher TIGIT expression in CLL compared to reactive LNs and ii) among CLL LNs a stronger TIGIT positivity in mutated vs unmutated cases, confirming flow cytometry data. In line with these findings, Richter's syndrome samples and patient-derived xenografts models showed the lowest TIGIT and the highest CD226 levels. We next examined TIGIT axis expression during the follow up of CLL cases who underwent treatment with BTK inhibitor (BTKi). While CD226 levels remained unmodified upon treatment, a sharp decrease in surface TIGIT was detected soon after BTKi initiation. Since TIGIT acts by decreasing TCR signaling to shut down T cell responses, we hypothesized similar functions in B cells. By crosslinking the BCR with an αIgM antibody in a selected cohort of IGHV UM CLL cells, we found that BTK phosphorylation was induced to a lesser extent in TIGIT high compared to TIGIT low samples, suggesting that TIGIT is a marker of CLL cell anergy. Accordingly, interruption of receptors/ligand interactions with RhTIGIT-Fc chimera or with αTIGIT or αCD226 blocking antibodies, modulated BCR signaling capacity. Specifically, in TIGIT high samples, preventing receptor engagement by CD155 increased αIgM-induced BTK phosphorylation; in contrast, in TIGIT low samples, blocking CD155 interaction affected mostly CD226 signaling, thereby depotentiating BCR activation. Similar results were obtained when stimulating CLL cells with CpG/IL-15. Interestingly, we observed a significant upregulation of surface CD226 in CLL cells cultured for 6 days in the presence of CpG/IL-15. CONCLUSIONS. These results show for the first-time expression of TIGIT by CLL cells. Furthermore, they indicate that TIGIT is a marker of CLL cells anergy, whereas activated CLL cells express high levels of CD226. Inhibition of TIGIT binding to CD155 partially restores B cell signaling and activation. Future studies are needed to gain insights on the mechanisms behind its deregulation and to obtain a complete functional characterization of the axis. Disclosures Coscia: AbbVie: Honoraria, Other; Janssen: Honoraria, Other, Research Funding; AstraZeneca: Honoraria; Gilead: Honoraria. Gaidano: Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astrazeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Allan: Genentech: Consultancy, Research Funding; Epizyme: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celegene: Research Funding; AstraZeneca Pharmaceuticals LP, Genentech, a member of the Roche Group, Janssen Biotech Inc, TG Therapeutics Inc.: Research Funding; AbbVie Inc, AstraZeneca Pharmaceuticals LP, BeiGene, Janssen Biotech Inc, Pharmacyclics LLC: Consultancy; AbbVie Inc, Ascentage Pharma, Epizyme, Genentech, a member of the Roche Group, Janssen Biotech Inc, Pharmacyclics LLC: Other: Advisory Committee; TG Therapeutics: Research Funding. Furman: Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy, Honoraria, Other: Expert testimony; Sunesis: Consultancy; Incyte: Consultancy; Beigene: Consultancy; Acerta/AstraZeneca: Consultancy; Loxo Oncology: Consultancy; Genentech: Consultancy; Morphosys: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; TG Therapeutics: Consultancy; X4 Pharmaceuticals: Consultancy; Janssen: Consultancy, Honoraria; AstraZeneca: Honoraria. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

Background: A rare complication of Chronic Lymphocytic Leukemia (CLL), Richter's Syndrome (RS) represents the transformation of a pre-existing CLL into a Diffuse Large B-cell Lymphoma (DLBCL), generally associated with poor prognosis. Current therapeutic approaches are limited and do not significantly reduce disease progression. For these reasons there is intense investigation to identify potentially druggable molecular circuits, opening the way to innovative combination therapies. Among the several oncogenic signalling pathways that may contribute to disease progression, the B Cell Receptor (BCR) is a main driver and an actionable target. We previously showed that BCR ligation in CLL cells increases expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage NAD pathway starting from nicotinamide, a finding in line with the notion of oncogenic-driven metabolic reprogramming. In this context, increased NAMPT expression leading to heightened NAD+ levels could sustain proliferation through the modulation of the activity of several intracellular enzymes, including sirtuins (NAD-dependent deacetylases). Aim: This work explores the connections between BCR signalling and the NAMPT/NAD/sirtuin axis in RS cells, asking the question of whether BCR and NAMPT can be simultaneously targeted. Methods: RS-PDX cells were freshly purified from tumor masses grown in mice and immediately used for short ex-vivo experiments. For in vivo experiments, NSG mice were injected subcutaneously with RS-PDX cells, which were left to engraft until a palpable mass was evident. Mice were then randomized to receive duvelisib, OT82 or a combination of the two for two consecutive weeks. Control mice were similarly treated with vehicle only. Mice were under a niacin-free diet. Results: By using our 4 RS patient-derived xenografts (PDX) models, we invariably observed high levels of NAMPT expression. High levels of NAMPT expression were also observed in 15 primary RS lymph node biopsies analyzed by RNA sequencing. BCR engagement through αIgM polyclonal antibodies significantly up-regulated NAMPT expression, as determined by qRT-PCR and protein analysis, with a concomitant increase in intracellular NAD+ levels. We then asked whether RS cells are sensitive to NAMPT inhibition, alone or in combination with drugs that target the BCR pathway. As most RS patients would likely have been treated during the preceding CLL phase with a BTK inhibitor, possibly developing resistance, we turned to PI3K inhibitors, which are less commonly used for CLL therapy. As NAMPT inhibitors (NAMPTi) we used both FK866 and OT-82, which are validated small molecules. Results indicated that the combination of the dual PI3K-δ/γ inhibitor duvelisib, with either FK866 or OT-82 induces dramatic apoptosis in all 4 models tested, as confirmed by annexinV/PI staining, by caspase 3 activation and by a significant drop in ATP and NAD+ levels. Importantly, two RS-PDX models (RS9737 and RS1316) were fully resistant to NAMPTi used alone, likely due to high levels of nicotinate phosphoribosyltransferase (NAPRT), which is the rate-limiting enzyme in the NAD salvage pathway that starts from nicotinic acid. However, addition of duvelisib, which was mildly effective when used alone, was followed by marked apoptosis even in these two models. Molecular dissection of the pathway showed that the combination of duvelisib and NAMPTi was followed by complete inhibition of the PI3K pathway, which was only partially blocked by duvelisib alone, even at high doses. The connection between NAMPT and PI3K is represented by cytoplasmic sirtuins, particularly SIRT2, which activate AKT through de-acetylation. Immunoprecipitation and two-dimensional gel electrophoresis showed that in the presence of NAMPTi, the amount of acetylated, i.e., inactive, AKT increased considerably. Consistently, treatment of RS-PDX mice with a combination of duvelisib and OT-82 was followed by significantly higher responses and longer animal survival. Conclusions: These results highlight a crosstalk between BCR signalling and NAMPT/sirtuin axis in RS models, showing the increased efficacy of the dual targeting (i.e., PI3K-δ/γ and NAMPT), and supporting this novel and promising therapeutic strategy for the treatment of RS patients. Disclosures Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

Representing the major cause of morbidity and mortality for chronic lymphocytic leukemia (CLL) patients, immunosuppression is a common feature of the disease. Effectors of the innate and the adaptive immune response show marked dysfunction and skewing towards the generation of a tolerant environment that favors disease expansion. Major deregulations are found in the T lymphocyte compartment, with inhibition of CD8+ cytotoxic and CD4+ activated effector T cells, replaced by exhausted and more tolerogenic subsets. Likewise, differentiation of monocytes towards a suppressive M2-like phenotype is induced at the expense of pro-inflammatory sub-populations. Thanks to their B-regulatory phenotype, leukemic cells play a central role in driving immunosuppression, progressively inhibiting immune responses. A number of signaling cascades triggered by soluble mediators and cell−cell contacts contribute to immunomodulation in CLL, fostered also by local environmental conditions, such as hypoxia and derived metabolic acidosis. Specifically, molecular pathways modulating T-cell activity in CLL, spanning from the best known cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death 1 (PD-1) to the emerging T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domains (TIGIT)/CD155 axes, are attracting increasing research interest and therapeutic relevance also in the CLL field. On the other hand, in the microenvironment, the B cell receptor (BCR), which is undoubtedly the master regulator of leukemic cell behavior, plays an important role in orchestrating immune responses, as well. Lastly, local conditions of hypoxia, typical of the lymphoid niche, have major effects both on CLL cells and on non-leukemic immune cells, partly mediated through adenosine signaling, for which novel specific inhibitors are currently under development. In summary, this review will provide an overview of the molecular and microenvironmental mechanisms that modify innate and adaptive immune responses of CLL patients, focusing attention on those that may have therapeutic implications.

Background: SARS-CoV-2 infection is heterogenous in clinical presentation and disease evolution. We investigated whether genetic variability can in of immune response to the virus can be influenced by genetic factors. Specifically, we compared HLA and AB0 frequencies in organ transplant recipients and in waitlisted patients according to the presence or absence of SARS-CoV-2 infection. Methods: A retrospective analysis was performed on an Italian cohort composed by subjects who received organ transplantation and candidates in waiting list in a January 2002-March 2020 time frame. Data from this cohort were merged with the Italian Ministry of Health registry of COVID+ subjects, evaluating infection status of transplanted and waitlisted patients. A total of 56304 cases were studied with the aim of comparing HLA and AB0 frequencies according to the presence (COVID+) or absence (COVID-) of SARS-CoV-2 infection. Findings: The cumulative incidence rate of COVID-19 was 0·112% in the Italian population and 0·462% in waitlisted/transplanted patients (OR=4·2, 95% CI [3·7-4·7], p

The systematic application of next-generation sequencing to large cohorts of oncologic samples has opened a Pandora's box full of known and novel genetic lesions implicated in different steps of cancer development and progression. Narrowing down to B cell malignancies, many previously unrecognized genes emerged as recurrently mutated. The challenge now is to determine how the mutation in a given gene affects the biology of the disease, paving the way to functional genomics studies. Mutations in NOTCH family members are shared by several disorders of the B series, even if with variable frequencies and mutational patterns. In silico predictions, revealed that mutations occurring in NOTCH receptors, despite being qualitatively different, may have similar effects on protein processing, ultimately leading to enhanced pathway activation. The discovery of mutations occurring also in downstream players, either potentiating positive signals or compromising negative regulators, indicates that multiple mechanisms in neoplastic B cells concur to activate NOTCH pathway. These findings are supported by results obtained in chronic lymphocytic leukemia and splenic marginal zone B cell lymphoma where deregulation of NOTCH signaling has been functionally characterized. The emerging picture confirms that NOTCH signaling is finely tuned in cell- and microenvironment-dependent ways. In B cell malignancies, it contributes to the regulation of proliferation, survival and migration. However, deeper biological studies are needed to pinpoint the contribution of NOTCH in the hierarchy of events driving B cells transformation, keeping in mind its role in normal B cells development. Because of its relevance in leukemia and lymphoma biology, the NOTCH pathway might represent an appealing therapeutic target: the next few years will tell whether this potential will be fulfilled.

ROR1 is a transmembrane receptor with tightly controlled expression during development. It is present on multiple tumor types but not on normal adult tissues. Hematological malignancies are often ROR1-positive, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and diffuse large B cell lymphoma (DLBCL). Given its unique pattern of expression, ROR1 represents a tumor-specific therapeutic target. The anti-ROR1 antibody, UC-961, is ahumanized IgG1 monoclonal antibody (mAb) that binds with high affinity to a specific extracellular epitope of human ROR1 receptor and can block Wnt5a-induced ROR1 signaling. Nonclinical studies document that UC-961 does not react with normal adult human tissues and selectively binds to tumor cells. Because of the antibody high specificity, rapid internalization, and trafficking to lysosomes, UC-961 appears ideally suited to serve as the targeting moiety for an anti-ROR1 ADC. Accordingly, we have developed VLS-101, a UC-961-linker-monomethyl auristatin E (MMAE) ADC that preserves the high-affinity binding and specificity of UC-961 and allows for ROR1-targeted intracellular release of MMAE. RS is an aggressive lymphoma, typically of DLBCL type, arising as transformation of CLL. Despite, progressive improvements in the therapy of CLL, very few effective treatment options exist for patients with RS. Using our recently established RS patient-derived xenografts (RS-PDXs), we explored the expression and signaling properties of ROR1 in RS and investigated the ex-vivo and in vivo effects of VLS-101. When assessed by flow cytometry (FACS), immunohistochemistry (IHC), and reverse-transcriptase-polymerase chain reaction (RT-PCR), 3 of 4 RS-PDXs showed ROR1 positivity (2 highly positive: 99% and 80% of cells; 1 medium/low positive: 25% of cells by FACS). The extent of ROR1 expression correlated among the 3 assays methods and was consistent with ROR1 expression data reported for non-RS DLBCL samples. When engaged by its ligand Wnt-5a, ROR1 activated downstream targets, Rac1 and RhoA, and induced phosphorylation of the p65 subunit of NF-kB and Jnk in RS cells. When cells purified from RS-PDX tumor masses were exposed to VLS-101 ex-vivo, the drug induced time- and dose-dependent apoptosis, as shown by increases in annexin V/propidium iodide and by Caspase-3 and PARP cleavage. VLS-101 efficacy was then assessed in vivo in both subcutaneous and systemic RS-PDX models. When palpable masses had formed in subcutaneous models, mice were randomly assigned to vehicle or VLS-101, bi-weekly treated for 3 consecutive weeks, and then compared for tumor growth and survival. In the 3 ROR1-expressing RS-PDX models, VLS-101 caused highly significant disease regressions. Complete regressions were observed even in RS-PDXs without universal ROR1 expression, suggesting a MMAE bystander effect. After treatment discontinuation, VLS-101-treated animals showed no tumor regrowth for several months and had significantly extended survival. Data were confirmed in systemic ROR1-expressing RS models in which tumor cells were intravenously injected and allowed to engraft for ~14 days before starting treatment. VLS-101 was administered with the same schedule adopted for the subcutaneous model. VLS-101 eliminated RS cells in bone marrow, peripheral blood, and spleen, increasing survival in VLS-101-treated animals relative to controls (approximately 20-50 days, depending on the RS-PDX model). Of note, no in vivo VLS-101 efficacy was observed in the ROR1-negative RS-PDX, confirming the specificity of VLS-101 targeting. VLS-101 showed no adverse effects on animal well-being or body weight. Collectively, these results indicate that ROR1 is expressed on RS cells where it can transduce pro-survival signals and can be diagnostically evaluated for selective targeting. Nonclinical data document that the MMAE-containing ADC, VLS-101, can cause RS-PDX apoptosis in vitro and can safely and selectively induce complete tumor regressions in in vivo models of RS derived from patient tumors with heavy prior clinical treatment and variable levels of ROR1 expression. Building on these types of results, a Phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with lymphoid cancers. Disclosures Vaisitti: VelosBio Inc.: Research Funding; Verastem Inc: Research Funding. Jessen:VelosBio Inc.: Employment. Vo:VelosBio Inc: Employment. Ko:VelosBio Inc: Employment. Allan:Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC, an AbbVie company: Consultancy; Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Bayer: Consultancy. Furman:Acerta Pharma: Consultancy; Pharmacyclics: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy; Incyte: Consultancy; Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Janssen: Consultancy. Miller:VelosBio Inc.: Employment. Lanutti:VelosBio Inc.: Employment. Deaglio:iTeos Therapeutics: Research Funding; Verastem Inc: Research Funding; VelosBio Inc.: Research Funding. OffLabel Disclosure: The drug used in this project is an anti-ROR1-toxin-conjugated antibody

Background The B cell receptor (BCR) is a signaling complex composed of surface immunoglobulin and heterodimer subunits, Igα (CD79a) and Igβ (CD79b) (Sanchez, et al. 1993). Chronic lymphocytic leukemia (CLL) demonstrates dim surface staining of CD79b compared to normal B cells or other B cell malignancies, in part due to overexpression of a splice variant that lacks coding for the extracellular domain (Alfarano, et al. 1999; Cabezudo, et al. 1999). Aggressive lymphomas, like diffuse large B cell lymphomas of non-germinal center origin (DLBCL-ABC), overexpress surface CD79b and can harbor gain of function mutations (Schmitz, et al. 2018). Richter's transformation (RT), a complication of CLL is defined by transformation to a DLBCL-ABC subtype most commonly. Upon transformation outcomes are poor and survival is short with standard therapeutic approaches. Polatuzumab vedotin (Pv), an antibody drug conjugate, targets CD79b on the surface and has obtained FDA approval for relapsed DLBCL in combination with bendamustine and rituximab. Targeting CD79b may represent an attractive therapeutic strategy for patients (pts) with RT. To this end we sought to characterize CD79b expression in RT. Methods Pts with CLL or RT and available paraffin embedded tissue blocks were identified in coordination with Weill Cornell Medicine's (WCM) pathology department. Clone AT107-2 (Bio-Rad, USA) which targets an intracellular epitope was used to stain for CD79b in formalin fixed paraffin embedded (FFPE) tissue sections after optimization following institutional staining procedures. CD79b was classified as either positive (pos) or negative (neg) based on staining pattern and intensity as deemed by a board certified hematopathologist. RT pt derived xenografts (RT-PDXs) were assessed for surface expression of CD79b via flow cytometry. Nonpermabilized cells derived from RT-PDXs were stained using an anti-human CD79b-FITC conjugated antibody (clone CD3-1, Southern Biotech, USA). Mean fluorescence intensity (MFI) was established. RNA seq data was obtained from 2 of 3 RT-PDX models and has been reported previously (Vaisitti, et al. 2018). CD79b transcripts per million (TPM) were analyzed and compared to that of normal lymph node (LN) tissue deposited in the Human Protein Atlas RNA Seq database (HPA-RNA Seq). GraphPad Prism 8.0 was used to perform statistical analysis. Results Nineteen pts with RT and 5 pts with CLL were identified for the study. Median age at diagnosis of the 19 RT pts was 71 years compared to 67 years for the 5 CLL pts. Five RT pts (26%) were treatment naïve (TN) at time of RT. Ten (53%) RT pts transformed on targeted therapy, 6 on BTK inhibitors, 2 on IMiD based therapy, 2 on venetoclax. CD79b stained pos for 16 RT pts (84%) and all 5 (100%) CLL pts. We were not able to identify any correlations between CD79b expression due to low numbers of neg cases. Surface expression of CD79b was evaluated on RT-PDX models from different passages and found to be pos in all 3 models with a median percentage of CD79b+ cells of 45.9% (range 34.6-76%). The median MFI was 2028.5 (range MFI 860-4289). Two of the 3 three models had bimodal populations of cells (pos and neg) whereas the third model was more uniformly pos. RNA seq data from 2 RT-PDX models was available and compared to RNA-seq data from normal LN tissue deposited in the HPA-RNA Seq database. The mean TPM of CD79b for RT-PDX cases was 837.5 compared to 311.5 for normal LN tissue, p=0.028. Conclusion CD79b expression was pos in 84% of FFPE primary RT specimens evaluated. We were able to confirm extracellular CD79b expression with flow cytometry in all 3 RT-PDX models with a median MFI of 2028.5. Surface expression was found to be bimodal in 2 models and uniformly pos in 1 model suggesting tumor heterogeneity. Rna-seq data from RT-PDX models demonstrated higher TPM values in RT-PDX samples compared to normal LN tissue. We conclude that RT expresses CD79b at sufficient levels despite deriving from a CLL cell that historically has demonstrated low surface CD79b expression. Further studies will be undertaken characterizing pos and neg cellular populations focusing on mechanisms of expression and potential differences in splice variant expression. Disclosures Allan: Pharmacyclics LLC, an AbbVie company: Consultancy; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Consultancy; Janssen: Consultancy, Honoraria; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Vaisitti:Verastem Inc: Research Funding; VelosBio Inc.: Research Funding. Joyce:Genentech: Employment. Schulz:Genentech, Inc.: Employment; Roche: Equity Ownership. Deaglio:Verastem Inc: Research Funding; iTeos Therapeutics: Research Funding; VelosBio Inc.: Research Funding. Furman:Genentech: Consultancy.

Richtersyndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, typically a diffuse large B cell lymphoma (DLBCL) with poor prognosis and without effective therapies. Chemotherapy, as well as novel inhibitory agents, show only partial and temporary responses, highlighting the need for alternative therapies that can overcome drug resistance. Duvelisib (DUV) is an oral phosphatidylinositol 3-kinase (PI3K)-δ and γ dual inhibitor showing efficacy in patients with refractory CLL. Venetoclax (VEN) is a Bcl-2 selective inhibitor, able to induce apoptosis in CLL cells. The lack of RS cell lines and murine models has limited understanding of the molecular mechanisms contributing to the pathogenesis of this disease, impacting also on the development of effective therapies. In our lab, 4 different RS patient-derived xenografts (PDXs) were established, representing useful tools to study the biology of the disease and to test novel therapeutic strategies. This work was undertaken to investigate expression and activity of the PI3K pathway and Bcl-2, and to test the ex-vivo and in vivo efficacy of DUV and VEN, alone or in combination, in our RS-PDX models. The 4 RS-PDX models are characterized by heterogeneous PI3K expression. RS1316 and IP867 expressed high levels of both p110δ and p110γ catalytic subunits. In contrast, RS9737 expressed low levels of both the PI3K catalytic subunits, while RS1050 expressed the p110δ subunit alone, but lacked p110γ and Bcl-2. We started by assessing the ex-vivo efficacy of DUV and VEN on RS-PDX-derived cells. Cells from the 4 RS-PDX models were obtained from disrupted tumor masses and immediately treated with DUV (5µM), VEN (25nM) or their combination. The DUV/VEN combination induced the highest levels of apoptosis in RS1316, RS9737 and IP867/17 cells at 24 and 48 h after treatment. Western blot confirmed the downregulation of Bcl-2 and Mcl-1, along with Caspase-3 and PARP-1 cleavage. In contrast, no effect was observed on RS1050 cells, which lack p110γ and Bcl-2, making this model intrinsically resistant to DUV and VEN treatment. Next, the effects of PI3K-δ/γ and Bcl-2 inhibition were examined in vivo on the RS1316-PDX model, selected as the best ex-vivo responder. NOD/SCID/gamma chain-/-(NSG) mice were subcutaneously injected with RS1316 cells. When tumor masses were palpable, animals were treated daily for 7 days including 2 holidays treatment with DUV (100 mg/kg p.o.), VEN (50 mg/kg p.o.) or their combination. In mice that received the combination, tumors were of smallest volume (4 out of 6 tumors were completely absent) and had the least viable cells, as highlighted by increment of cleaved Caspase-3 immunohistochemistry signal. In a second set of experiments, tumor masses were left to grow after 10 consecutive treatments and monitored daily. Used as single agents, DUV and VEN significantly reduced tumor growth, prolonging survival for 5 days compared to controls (mean survival 42 days in the control condition vs 47 in the single treatments). The combination of the two drugs was even more efficacious, leading to a complete remission in all the mice during treatment (Fig. 1). Tumor masses eventually grew back after cessation of treatment, but the combination significantly prolonged survival compared to vehicle or single-treatment groups (mean survival 56 days, p In conclusion, DUV and VEN treatment combination, simultaneously inhibiting PI3K-δ/γ and Bcl-2, induced RS tumor regression, offering a promising treatment combination, at least for RS patients expressing high levels of PI3K gamma and delta subunits and Bcl-2. These results pave the way for clinical testing of duvelisib and venetoclax combination for patients with RS. Figure 1 Disclosures Furman: AstraZeneca: Consultancy; Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy; Acerta Pharma: Consultancy; Janssen: Consultancy; Incyte: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Beigene: Consultancy; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy. Allan:Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC, an AbbVie company: Consultancy; Janssen: Consultancy, Honoraria; Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coma:Verastem Inc: Employment. Pachter:Verastem Inc: Employment. Deaglio:VelosBio Inc.: Research Funding; iTeos Therapeutics: Research Funding; Verastem Inc: Research Funding. Vaisitti:Verastem Inc: Research Funding; VelosBio Inc.: Research Funding. OffLabel Disclosure: Duvelisib: PI3K gamma and delta inhibitor Venetoclax: bcl2 inhibitor

Human CD38, an ecto-enzyme and a receptor, performs as an independent negative prognostic marker for patients with chronic lymphocytic leukemia (CLL), a hematological malignancy characterized by the accumulation of a population of mature B lymphocytes expressing CD5. Patients with a CD38 + CLL clone display a more aggressive form of the disease with earlier treatment requirements and ultimately shorter overall survival than patients with a CD38 - clone. Several lines of evidence indicate that CD38 is not only a diagnostic marker but also a key element in the molecular network regulating disease maintenance and progression. First, CD38 is a receptor that induces proliferation and increases survival of CLL cells. Second, CD38 signals facilitate access of CLL cells to growth-favorable districts. This is achieved by enhancing i) chemotaxis towards CXCL12, ii) integrin-mediated adhesion and iii) matrix metalloprotease synthesis and secretion. Third, blocking monoclonal antibodies targeting CD38 impair CLL homing to spleen and bone marrow in xenograft models. These functions appear to be modulated by frontal interactions with CD31 as well as by lateral associations on the CLL membrane to form a large supramolecular complex similar to the invadosomes of epithelial cells. Our understanding has evolved from considering CD38 as a marker of unfavorable prognosis to recognizing its function as a disease modifier. Studies in the next few years will likely determine whether the molecule can also serve as a target for new therapies, using monoclonal antibodies, inhibitors of the enzymatic activity or both.

Extracellular adenosine (ADO), generated from ATP or ADP through the concerted action of the ectoenzymes CD39 and CD73, elicits autocrine and paracrine effects mediated by type 1 purinergic receptors. We have tested whether the expression of CD39 and CD73 by chronic lymphocytic leukemia (CLL) cells activates an adenosinergic axis affecting growth and survival. By immunohistochemistry, CD39 is widely expressed in CLL lymph nodes, whereas CD73 is restricted to proliferation centers. CD73 expression is highest on Ki-67+ CLL cells, adjacent to T lymphocytes, and is further localized to perivascular areas. CD39+/CD73+ CLL cells generate ADO from ADP in a time- and concentration-dependent manner. In peripheral blood, CD73 expression occurs in 97/299 (32%) CLL patients and pairs with CD38 and ZAP-70 expression. CD73-generated extracellular ADO activates type 1 purinergic A2A receptors that are constitutively expressed by CLL cells and that are further elevated in proliferating neoplastic cells. Activation of the ADO receptors increases cytoplasmic cAMP levels, inhibiting chemotaxis and limiting spontaneous drug-induced apoptosis of CLL cells. These data are consistent with the existence of an autocrine adenosinergic loop, and support engraftment of leukemic cells in growth-favorable niches, while simultaneously protecting from the action of chemotherapeutic agents.

CD38, a nucleotide-metabolizing ectoenzyme and a receptor, is a negative prognostic marker for chronic lymphocytic leukemia (CLL) patients. CD38 has a genetic polymorphism, with a C → G variation in a putative E-box located in a regulatory region. E2A, the predominant E-box factor in B lymphocytes, was found to be highly expressed by CD38(+) CLL patients. The highest CD38 levels scored by E2A(+)/G carrier patients suggested that E2A is (i) directly associated with CD38 expression, and that (ii) the binding of the transcription factor is influenced by the CD38 genotype. Chromatin immunoprecipitation indicated that E2A directly interacts with the CD38 regulatory region. Furthermore, E2A binding was stronger in the presence of the G allele. Experiments of E2A silencing led to a significant reduction of surface levels of CD38, confirming the working hypothesis. A direct functional interplay between E2A and CD38 was shown by exposing CLL cells to interleukin-2 and TLR-9 ligands, both inducers of CD38 expression. Under these conditions, CD38 upregulation was primarily conditioned by the presence of E2A and then by the G allele. The results of this study link E2A and CD38 expression within a common pathway, in which E-protein activity is required for the efficient induction of CD38 transcription.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukaemia (CLL) to aggressive lymphoma. Risk factors of CLL transformation to RS are only partly known. We explored the role of the host genetic background as a risk factor for RS occurrence. Forty-five single nucleotide polimorphisms (SNPs) known to be relevant for CLL prognosis were genotyped in a consecutive cohort of 331 CLL, of which 21 had transformed to RS. After correcting for multiple testing and adjusting for previously reported RS risk factors, the LRP4 rs2306029 TT variant genotype was the sole SNP independently associated with a higher risk of RS transformation (Hazard Ratio: 4·17; P = 0·001; q = 0·047). The enrichment of LRP4 TT genotype in RS was confirmed in an independent series (n = 44) used for validation purposes. The LRP4 protein was expressed in CLL (n =66). Bioinformatic analysis scored LRP4 rs2306029 as a variant with possible deleterious and damaging variant of LRP4. LRP4 genotyping may help the recognition of patients with increased risk of RS at the time of CLL diagnosis.

CLL is characterized by a dynamic balance between cells proliferating in the lymphoid organs and circulating cells resisting programmed cell death. Regulating this equilibrium entails complex interactions between tumor and host, modulated by a set of surface molecules expressed by the CLL cell according to environmental conditions. The result is a constantly shifting pattern of resistance, apoptosis and proliferation. The CD38 surface molecule is an independent negative prognostic factor expressed by approximately one-third of CLL patients. Our view is that CD38 is crucial to tumor-host communication and that its signals are detrimental to clinical outcome. CD38(+) CLL cells can proliferate in vitro in the presence of anti-CD38 mAbs and IL-2 and are more sensitive to the effects of the CXCL12 chemokine. Blockage of CD38 signals impairs CLL cell movement from blood to lymphoid organs, as confirmed using animal models. One model to be explored considers CD38 a key component of the CLL invadosome, a still hypothetical membrane domain containing adhesion molecules, chemokine receptors and matrix metalloproteases. Some components of the invadosome are genetically polymorphic, explaining heterogeneity in functional response. The CD38 gene shows genetic differences in the promoter region, some of which represent an independent risk for Richter transformation. In addition to driving the clinical outcome of the disease, CD38 is thus an excellent candidate therapeutic target for a significant subset of CLL patients.

Homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth, a critical step in disease progression, is principally coordinated by the CXCL12/CXCR4 axis. A cohort of 62 CLL patients was divided into migrating and nonmigrating subsets according to chemotaxis toward CXCL12. Migrating patients phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) proteins more than nonmigrating patients (P0.0002). CD38 expression was the parameter most strongly associated with heightened CXCL12 signaling (P0.0001), confirmed by independent statistical approaches. Consistent with this observation, CD38(-) CLL cells in samples with bimodal CD38 expression responded less to CXCL12 than the intact clone (P=0.003). Furthermore, lentivirus-induced de novo expression of CD38 was paralleled by increased responses to CXCL12, as compared with cells infected with a control virus. CD38 ligation with agonistic monoclonal antibodies (mAbs) enhanced CXCL12 signaling, whereas blocking anti-CD38 mAbs inhibited chemokine effects in vitro. This is attributed to physical proximity on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by (i) coimmunoprecipitation and (ii) confocal microscopy experiments. Blocking anti-CD38 mAbs significantly compromised homing of CLL cells from blood to lymphoid organs in a mouse model. These results indicate that CD38 synergizes with the CXCR4 pathway and support the working hypothesis that migration is a central step in disease progression.

Our interest in chronic lymphocytic leukemia (CLL) derives primarily from the exploitation of human diseases as strategic models for defining the in vivo biological roles of CD38. Using this model, we showed that CD38 triggers robust proliferation/survival signals modulated through the interactions with the CD31 ligand expressed by nurselike cells and by the stromal/endothelial components. By analyzing a cohort of 56 patients with clinically and molecularly characterized CLL, we show that (1) patients with CD38+/ZAP-70+ are characterized by enhanced migration toward Stromal derived factor-1α (SDF-1α)/CXCL12; (2) CD38 ligation leads to tyrosine phosphorylation of ZAP-70, showing that these markers are functionally linked; (3) ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, as shown by studying CD38-mediated signal transduction in 26 molecularly characterized patients; and (4) the CLL subgroup of patients defined on the basis of migratory potential is marked by a specific genetic signature, with a significant number of differentially expressed genes being involved in cell-cell interactions and movement. Altogether, the results of this work provide biological evidence for why the combined analysis of CD38 and ZAP-70 expression as determined in several clinical trials results in more dependable identification of patients with CLL who have aggressive disease.

Chronic lymphocytic leukemia (CLL) is a variable disease; therefore, markers to identify aggressive forms are essential for patient management. Here, we have shown that expression of the costimulatory molecule and microbial sensor SLAMF1 (also known as CD150) is lost in a subset of patients with an aggressive CLL that associates with a shorter time to first treatment and reduced overall survival. SLAMF1 silencing in CLL-like Mec-1 cells, which constitutively express SLAMF1, modulated pathways related to cell migration, cytoskeletal organization, and intracellular vesicle formation and recirculation. SLAMF1 deficiency associated with increased expression of CXCR4, CD38, and CD44, thereby positively affecting chemotactic responses to CXCL12. SLAMF1 ligation with an agonistic monoclonal antibody increased ROS accumulation and induced phosphorylation of p38, JNK1/2, and BCL2, thereby promoting the autophagic flux. Beclin1 dissociated from BCL2 in response to SLAMF1 ligation, resulting in formation of the autophagy macrocomplex, which contains SLAMF1, beclin1, and the enzyme VPS34. Accordingly, SLAMF1-silenced cells or SLAMF1(lo) primary CLL cells were resistant to autophagy-activating therapeutic agents, such as fludarabine and the BCL2 homology domain 3 mimetic ABT-737. Together, these results indicate that loss of SLAMF1 expression in CLL modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, and suggest that these effects underlie unfavorable clinical outcome experienced by SLAMF1(lo) patients.

El artículo está disponible gratuitamente en la web de la editorial, This paper reviews some of the results and the speculations presented at the Torino CD38 Meeting in June, 2006 and focused on CD38 and CD157 seen as a family of molecules acting as surface receptors of immune cells. This partisan view was adopted in the attempt to combine the enzymatic functions with what the immunologists consider key functions in different cell models. At the moment, it is unclear whether the two functions are correlated, indifferent, or independent. Here we present conclusions inferred exclusively on human cell models, namely T and B lymphocytes, dendritic cells, and granulocytes. As an extra analytical tool, we try to follow in the history of life when the enzymatic and receptorial functions were generated, mixing ontogeny, membrane localization, and cell anchorage.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.

BACKGROUND: Tumor cell survival critically depends on heterotypic communications with non-malignant cells in the microenvironment. Most of these signals converge on the activation of the transcription factor NF-κB that regulates complex cellular functions, including apoptosis, cell survival and proliferation. Even if NF-kB is constitutively active in most malignancies, including chronic lymphocytic leukemia (CLL), and plays a major role in tumorigenesis, there are no currently approved drugs to target it. IT901 has been recently reported as a novel NF-kB inhibitor, showing efficacy in a non-tumor context1. AIM OF THE WORK: The aim of this work is to test the efficacy of IT901 in CLL and in its more aggressive transformation, Richter syndrome (RS), which represents an unmet therapeutic need. The molecular mechanisms of action of IT901 in leukemic cells are studied, alongside its effects on cells belonging to CLL microenvironment. RESULTS: IT901 induces apoptosis in primary leukemic cells in a dose- and time-dependent manner, showing significant efficacy after 24h of treatment. The apoptotic response is independent of the prognostic subgroup. Conversely, IT901 has minimal impact upon normal B cells. Treatment of CLL cells with IT901 interferes with NF-kB transcriptional activity, resulting in a diminished binding of both p50 and p65 to DNA. Moreover, biochemical analyses indicate a diminished expression of these subunits in the nucleus, as well as of the whole NF-kB complex in the cytoplasm. At the molecular level, compromised expression of NF-kB triggers activation of the Caspase-3 apoptotic pathway, with increased expression of pro-apoptotic proteins (e.g., Bim), paralleled by a diminished expression of the anti-apoptotic ones (e.g., XIAP). Concomitantly, a prominent increase in mitochondrial ROS is evident, providing a link between IT901 effects and induction of apoptosis. Recent data reported the involvement of NF-kB as a transcriptional controller of metabolic pathways promoting oxidative phosphorylation in cancer cells. In line with NF-kB constitutive activation in CLL, dynamic measurement of the energetic profile, indicates a reliance on oxidative phosphorylation, with limited glycolytic capacity. After IT901 treatment, there is a dramatic drop in mitochondrial respiration, with compromised ATP production and a net increase in proton leak, suggesting that primary CLL cells are trying to compensate impaired respiration by shifting to glycolysis. This metabolic response is mediated at the transcriptional levels, as IT901 induces a down-modulation of the genes involved in mitochondrial respiration (e.g., ATP5A1) and a concomitant up-modulation of the ones involved in glucose uptake and lactate transport (e.g., GLUT1). The CLL microenvironment is critical for disease progression and for providing protection from drug-induced apoptosis. Therefore it is important to consider the effects of novel drugs also on non-neoplastic bystander elements. Nurse-like cells (NLC) are a population of monocyte-derived activated macrophages that nurtures CLL cells via soluble and cell contact dependent mechanisms. These interactions are known to activate NF-kB signaling in both partners. Consistently, IT901 inhibited nuclear localization of the p65 subunit in NLC and shifted their polarization towards an M1-phenotype. These results are confirmed using a xenograft model. The Mec-1 cell line was injected into NSG mice and left to engraft for 2 weeks before beginning treatment. Animals treated with IT901 are characterized by decreased tumor growth and leukemic cells diffusion compared to controls, as shown by a diminished number of leukemic cells in kidneys, liver and spleen. Finally, IT901 shows promising effects in a small cohort of leukemic cells obtained from RS patients, inducing significant apoptosis by interfering with the expression and nuclear localization of NF-kB. CONCLUSIONS: Altogether, these results indicate that IT901 blocks NF-kB transcriptional activity. This effect is followed by rapid and marked decrease in genes supporting oxidative phosphorylation, causing mitochondrial damage, ROS release and induction of intrinsic apoptosis. Moreover, IT901 interrupts the support that CLL obtains from the microenvironment. Thus, targeting NF-kB by means of IT901 may be effective for CLL, and possibly even RS patients. 1. Y. Shono et al., Cancer Res76, 377 (Jan 15, 2016). Disclosures Furman: Genentech: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau.

BACKGROUND . NOTCH1 PEST domain mutations were among the first to be identified using whole exome sequencing and their prevalence and prognostic power for CLL patients has since been extensively studied. Nevertheless, the functional contribution of NOTCH1 and the impact of its mutations in CLL remain poorly understood. This is mainly due to the intrinsic limits in using primary CLL cells and to the rapid inactivation of the NOTCH1 pathway in vitro. AIM. The aim of the study was to highlight the NOTCH1-dependent mechanisms contributing to CLL pathogenesis and progression and the effects of PEST mutations in this disease context. RESULTS. By using the CRISPR/Cas9 technology, we first generated a cellular model either lacking NOTCH1 (Mec-1/KO) or expressing it in its wild-type or mutated forms. RNA-seq analysis of the transcriptome of Mec-1/WT and /KO cells, highlighted signaling and migration as the most prominently down-regulated pathways in KO cells. These findings were confirmed by showing that Mec-1/KO cells migrated significantly less towards CCL19 than their WT counterparts, as the result of the down-modulation of CCR7, the CCL19 receptor. Mechanistically, Mec-1/KO were characterized by increased expression of the tumor suppressor gene DUSP22, a phosphatase that negatively regulates MAPK and STAT3 signaling, in turn responsible for CCR7 gene transcription. Up-regulation of DUSP22 in Mec-1/KO was caused by decreased methylation of the gene promoter, as shown using a methylation specific PCR. Re-expression of the NOTCH1 Intracellular Domain (NICD) rescued the phenotype, increasing methylation of the DUSP22 promoter, which in turn led to decreased gene and protein expression. Mec-1 cells with reconstituted NICD expression showed increased STAT3 phosphorylation, CCR7 expression and, ultimately, migrated more actively in response to CCL19. Importantly, reconstitution with an NICD carrying the PEST domain mutation most commonly found in patients (ΔCT_7541-7542), significantly enhanced all these events, including DUSP22methylation, STAT3 phosphorylation, CCR7 expression, generating cells with the highest chemotactic responses to CCL19. NOTCH1 directly participates to the epigenetic regulation of DUSP22, by conditioning the activity of the DNA methyltransferase 3A (DNMT3A) on the gene promoter. By binding RBPJk, the NICD displaces HDAC1 from the repressor complex and initiates NOTCH1-dependent signaling. Consequently, free HDAC1 binds to and stabilizes DNMT3A, promoting its activity on DUSP22promoter. These results were first corroborated in a large cohort of CLL (n=113) carrying mutations in NOTCH1 PEST domain (ΔCT_7541-7542)at the clonal (>12%) or subclonal (≤12%) level. The clonally mutated subset showed significant downregulation of DUSP22expression and gene promoter hypermethylation compared to subclonal samples. Consistently, the clonal subset displayed higher constitutive STAT3 phosphorylation, expressed higher levels of CCR7 and migrated more efficiently to CCL19 than subclonally mutated CLL. The second confirmation was obtained by studying a Mec-1 variant with PEST domain mutation (Mec-1/PEST), generated using the CRISPR/Cas9 system. Like primary cells, Mec-1/PEST needed the ligand to initiate signaling. As predicted, the mutant NICD was more stable and more transcriptionally active. Consistently, these cells had the lowest DUSP22 levels and the highest CCR7 expression. When Mec-1/PEST were xenografted in immunocompromised mice, activation of the NOTCH1 pathway was more pronounced than that of Mec-1/WT cells. In keeping with their minimal expression of DUSP22, Mec-1/PEST cells were characterized by markedly increased metastatic properties, with extensive colonization of the liver, the spleen, and the brain, at variance with the other cell variants. CONCLUSIONS . Considered together, these results show that PEST mutations increase NICD stability, in turn affecting a complex nuclear balance. The final outcome for the CLL cell is decreased expression of the tumor suppressor gene DUSP22 and increased chemotaxis towards CCL19. As this chemokine regulates homing to secondary organs, conceivably NOTCH1 mutations might favor CLL recirculation to lymph node and spleen, where the local environment triggers proliferation and protects from apoptosis, two conditions that are associated with a more aggressive disease and an unfavorable prognosis. Disclosures Coscia: Karyopharm: Research Funding; Janssen: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; ROCHE: Honoraria, Other: Advisory board. Furman:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria.

The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.

Because of its relatively indolent clinical course, chronic lymphocytic leukemia (CLL) offers a versatile model for testing novel therapeutic regimens and drug combinations. Nicotinamide is the main NAD+ precursor and a direct inhibitor of four classes of enzymes, including the sirtuins. SIRT1, the main member of the sirtuin family, inactivates p53 by deacetylating a critical lysine residue. In this study, we showed that CLL cells express high levels of functional SIRT1, which is inhibited by exogenous nicotinamide. This agent blocks proliferation and promotes apoptosis selectively in leukemic cells that express wild-type (wt) p53. Nicotinamide modulates the p53-dependent genes p21, NOXA, BAX, and Mcl-1, indicating an activation of the p53 pathway and of caspase-3. DNA-damaging chemotherapeutics, such as etoposide, activate a functional loop linking SIRT1 and p53 through the induction of miR-34a. When leukemic cells are simultaneously exposed to nicotinamide and etoposide, we observe a significant increase in miR-34a levels with a concomitant inhibition of SIRT1. Furthermore, p53 acetylation levels are higher than with either agent used alone. Overall, treatment with both nicotinamde and etoposide shows strongly synergistic effects in the induction of apoptosis. We therefore concluded that nicotinamide has the dual property of inhibiting SIRT1 through a noncompetitive enzymatic block (p53 independent) and at the same time through miR-34a induction (p53 dependent). These observations suggested the therapeutic potential of nicotinamide, a novel, safe, and inexpensive drug, to be used in addition to chemotherapy for CLL patients with wt p53. Cancer Res; 71(13); 4473–83. ©2011 AACR.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme that can be released in the extracellular milieu. Here, it may elicit signals through binding purinergic receptors. Alternatively, NAD+ may be dismantled to adenosine, up-taken by cells and transformed to reconstitute the intracellular nucleotide pool. An articulated ecto-enzyme network is responsible for the nucleotide–nucleoside conversion. CD38 is the main mammalian enzyme that hydrolyzes NAD+, generating Ca2+-active metabolites. Evidence suggests that this extracellular network may be altered or used by tumor cells to (i) nestle in protected areas, and (ii) evade the immune response. We have exploited chronic lymphocytic leukemia as a model to test the role of the ecto-enzyme network, starting by analyzing the individual elements that make up the whole picture.

Extracellular adenosine generated from ATP/ADP through the concerted action of the ectoenzymes CD39 and CD73 elicits potent cytoprotective and immunosuppressive effects mediated by type-1 purinergic receptors. Chronic lymphocytic leukemia (CLL) patients expressing the ectoenzymes CD39 and CD73 can actively produce adenosine. This condition activates an autocrine adenosinergic axis that supports engraftment of leukemic cells in a growth-favorable environment. These effects are mediated by the A2A adenosine receptor, which inhibits chemotaxis and limits spontaneous and drug-induced apoptosis of CLL cells. Following the reported cross-talk between hypoxia and adenosine, we tested the hypothesis of a functional interplay between the adenosinergic axis and hypoxic signals in the CLL microenvironment. Results confirmed that CLL cells robustly increase HIF-1α expression when cultured under hypoxia. Under these conditions a significant increase in the mRNA and protein levels of CD73, CD26 and A2A was observed. An HPLC assay confirmed that hypoxic CLL cell cultures produce higher extracellular adenosine levels compared to normoxia. The increase is apparent upon inhibition of adenosine deaminase and nucleoside transporters, suggesting that under hypoxia scavenging mechanisms are activated. Attention was then focused on the stromal and T cell compartments, which are critical to the formation and maintenance of the leukemic niche. Hypoxia enhanced differentiation of circulating monocytes into nurse-like cells, macrophages of the M2 type playing an essential role in nurturing leukemic cells. During hypoxia, differentiated NLC overexpressed A2A, which was also functional. Indeed, an increased AKT and ERK1/2 phosphorylation was observed following pharmacological activation. The enhancement of NLC differentiation under hypoxic conditions relied, at least in part, on the activation of A2A: its engagement by the agonist favored NLC generation in normoxia, with overexpression of IDO, CD163 and CD206. Moreover, activation of A2A induced secretion of immunomodulatory cytokines, such as IL-6, IL-10 and CCL18, while pharmacological blockade of A2A under hypoxia prevented NLC growth. In the T cell compartment, hypoxic cultures were followed by the sharp up-regulation of the A2A, without significantly affecting the enzymes that generate adenosine. Co-cultures of T lymphocytes and CLL cells under hypoxia resulted in a dramatic decrease of T cell proliferation, partially rescued by antagonists of A2A. Furthermore, expression of the PD-1 immunoinhibitory receptor was enhanced in hypoxic T cells, suggesting that there are multiple inhibitory mechanisms. Together, these results indicate that the adenosinergic and hypoxic axes synergize in shaping the CLL niche, suggesting that the pharmacological inhibition of adenosinergic signals may counteract some of the effects mediated by an hypoxic environment. Citation Format: Sara Serra, Davide Brusa, Roberta Buonincontri, Valentina Audrito, Tiziana Vaisitti, Simon Robson, Silvia Deaglio. Cooperation between adenosinergic and hypoxic signals in shaping chronic lymphocytic leukemia microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3192. doi:10.1158/1538-7445.AM2015-3192