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Chronic lymphocytic leukemia (CLL) is characterized by low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL to an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1- mutated CLL cells, including a gene expression profile enriched in NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and downregulation of surface CD20 in SF3B1-mutated CLL cells correlate with overexpression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings were confirmed by separately analyzing the CD20dim and CD20bright cell fractions from SF3B1-mutated cases as well as by DVL2 knockout experiments in CLL-like cell models. Together, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding therapies based on novel agents to SF3B1-mutated CLL.

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Context: Ibrutinib inhibits BTK with the most typical response in CLL being partial remission (PR) with measurable minimal residual disease (MRD) in blood. Remission is usually maintained until development of genetically-driven resistance. Objective: Mechanism of adaptation and survival in MRD. Patients and Methods: Pre-treatment CLL cells and under-treatment MRD were systematically collected from 33 high-risk CLL patients. Samples were characterized by high-dimensional, single-cell flow cytometry; bulk genomic, transcriptomic, and chromatin accessibility profiling; and single-cell RNA-seq profiling. Results: The genetic composition of MRD remained constant across timepoints, indicating that ibrutinib did not have any impact in shaping its genomic diversity. Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib were enriched of binding sites for transcription factor that lay downstream ERK signaling. Regions that turned into a less accessible state were enriched for the binding sites of TF emanating from the BCR. At the transcriptomic level, most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD, while only a fraction was upregulated. IL4, NF-kB, and metabolism and proliferation signatures were downregulated in MRD, while MAPK and RAS signatures were upregulated. CLL spleen samples from TCL1 mice under ibrutinib treatment showed an upregulation of MAPK pathway genes compared to untreated mice. Single-cell transcriptomes revealed a distinct post-treatment cell population driven by MAPK activity that functionally pre-existed in a fraction of CLL cells of baseline samples before ibrutinib start. At the signaling level, ibrutinib had no effect on the integrity of RAS-BRAF-MAPK-ERK pathway protein expression upon crosslinking of the BCR with anti-IgM but not after stimulation of TLR9 or CD40. Functional validation on primary samples by western blotting shows that RAS/ERK can be activated by BCR stimulation, irrespective of ibrutinib treatment in ex vivo experiments. Pharmacological inhibition of MEK (trametinib) and ERK (ulixertinib) synergized with ibrutinib, leading to decreased cell viability. Conclusions: MRD under ibrutinib adapts its phenotype in an epigenetic way to maintain functional competence of BCR signaling via the MAPK pathway, which turned to be a vulnerability of MRD persisting under ibrutinib.

Significance CLL is characterized by autonomous B cell receptor (BCR) signaling. CLL subsets are empirically defined by sequence similarities of the BCR heavy chain. However, in the unfavorable subset 2, an acquired mutation (termed R110) in the light chain stimulates autonomous BCR signaling. This study demonstrates that the oncogenic R110 mutation dictates the unfavorable prognosis and is not restricted to the conventional subset 2. Interestingly, carriers of a particular light-chain allele (IGLV3-21*01) are predisposed to develop CLL because this allele enables autonomous BCR signaling by R110 as a single-point mutation. Monoclonal antibodies permit convenient screening for R110-expressing CLL, showing that it is the largest immunologically defined CLL subset and an example of functional rather than empirical CLL subclassification., The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21–derived heavy chains (HCs) with IGLV3-21–derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110–expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR–BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01–expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110–expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by gamma-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541- 7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC- mediated epigenetic repression of CD20 expression.

In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10–15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (Po0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. Especially, a continuous BCR signaling, which is either induced by canonical autoantigen-dependent mechanism, or by an autonomous manner, may augment VLA-4 activation. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this feature with the presence of signals from the BCR. Methods. Constitutive VLA-4 activation was determined in flow cytometry by combining expression of CD49d and activated CD29, the latter using the conformation-sensitive anti-CD29 mAb HUTS21 (Tissino et al, J Exp Med, 2018) in whole blood (WB), as source of VLA-4 ligands, from: i) 1,044 consecutive CLL all with IGHV gene mutations available; ii) sequential samples (0, 14, 30, 90 days) from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=15) and from a clinical trial (NCT02827617, n=15). HUTS21 staining was also performed in the presence of: i) plasma depletion/replacement; ii) soluble (s) VCAM-1; iii) fibronectin (FN); iv) blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). Antigene driven BCR signaling and autonomous BCR signaling were investigated by Ca++ influx assay in a 4-hydroxy tamoxifen (4-OHT) inducible manner in murine TKO cells system as described before (Dühren-von Minden M et al, Nature, 2012). VLA-4 activation was assessed by "real-time" flow cytometry measuring the binding of the VLA-4 ligand LDV-FITC and its replacement by unlabeled LDV. The calculated Koff values indicate the VLA-4 affinity state (Koff 0.06 s−1 low affinity). BCR engagement was performed using goat F(ab′)2 anti-human IgM. Results. 1) A fraction of CD49d+ CLL shows constitutive VLA-4 activation - Out of 1,044 CLL, 534 (51%) expressed CD49d (cutoff 30%), and HUTS21 (cutoff 20%) was scored positive in 112/534 (21%) cases. HUTS21 staining was: i) impaired by depletion of plasma from WB samples (Fig.A), and reconstituted by specific plasma components (sVCAM1, FN; Fig.B); ii) impaired by pre-incubation with anti-CD49d HP2/1 blocking mAbs before addition of plasma, sVCAM-1 and FN (Fig.C). 2) Plasma levels of sVCAM-1 are dependent on the VLA-4 activation state - sVCAM-1 (n=122 plasma samples) was higher in CD49d+ vs CD49d- cases (p 3) Constitutive VLA-4 activation associates to specific BCR - By comparing cases expressing high vs low levels of constitutively activated VLA-4 (HUTS21>50% vs HUTS21 4) Constitutive VLA-4 activation is hampered by IB exposure in-vivo - A decrease in constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30 and 90 from patients undergoing IB treatment (Fig.F), suggesting an IB-dependent impairment of VLA-4 activation via BCR signal. 5) VLA-4 is activated via BCR autonomous signaling - Murine TKO cells express high level of cell surface VLA-4 (Fig.G). TKO cells expressing different BCRs derived from CLL cases (e.g. BCR17, a CLL- subset#2 case) show autonomous Ca++ influx as well as VLA-4 activation as compared to TKO cells expressing BCRs derived from healthy donor (BCR53, mature recirculating B cells). Notably, both BCR17 and BCR53, upon anti-IgM stimulation, induced high VLA-4 affinity state, while only BCR17 induced a significant increase of VLA-4 affinity in the absence of anti-IgM stimulation (Fig.H). Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a significant fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling. Figure Disclosures Di Raimondo: Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board.

Trisomy 12 is a recurrent cytogenetic abnormality that occurs in 15–20% of Chronic Lymphocytic Leukemia (CLL).[1][1],[2][2] Within the hierarchical model proposed by Dohner et al .,[3][3] trisomy 12 CLL (tris12 CLL) carry an intermediate prognostic risk, with median overall survival (OS) and time

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of tumor cell-microenvironment interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its specific ligands. In this context, VLA-4 needs to be maintained in an activated state by a continuous stream of inside-out signals from the BCR, which can be triggered by canonical antigens as well as in an autonomous antigen-independent manner, a BCR-mediated signaling recently emphasized to specifically occur in CLL cells. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this still not described feature with the presence of signals from the BCR continuously activating VLA-4. Methods. Expression of the integrin alpha (CD49c, CD49d, CD49e) and beta1 (CD29) chains was analyzed by flow cytometry. The VLA-4 activation state was investigated by flow cytometry using the conformational sensitive anti-CD29 monoclonal antibody HUTS21, employed in conjunction with sources of VLA-4 ligands: either autologous plasma-containing fibronectin (FN) and soluble (s) VCAM-1 or increasing concentrations of exogenously added LDV-containing peptides as a VLA-4 specific ligand. In detail: i) HUTS21 expression was analyzed using whole blood (WB) samples from 727 CLL patients. Negative controls were obtained after plasma depletion through washing of WB samples; ii) the VLA-4 activation state expressed as receptor occupancy (RO) (J Biol Chem, 284,14337, 2009) was analyzed in sequential (t=0, day14, day 30) frozen/thawed samples from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=16) and from a clinical trial (NCT02827617, n=14). BCR engagement was performed using goat F(ab′)2 anti-human IgM. ELISA assays were used to quantify FN and sVCAM-1 in plasma samples. Results. According to the 30% cutoff, CD49d was expressed in 351/727 (48%), while CD29 was expressed in 676/727 (93%), and a strong correlation between CD49d and CD29 was observed (rho=0.7, p20% (arbitrary cut-off) HUTS21 expression. Notably, HUTS21 positive expression was not circumscribed to VLA-4 expressing CLL, being also detected in 64/375 (17%) CD49d-, CD49c+ and/or CD49e+ CLL, thus envisioning a role of other integrins (CD49c and CD49e) in CLL cell interactions in tissues sites. HUTS21 expression did not correlate with a definite cytogenetic group, IGHV mutational status or with a specific IGHV family and stereotype. Depletion of the plasma component from the WB samples before HUTS21 staining significantly reduced the proportion of HUTS21 positive cells compared with those measured in WB samples (p=0.001, n=11), suggesting the need of plasma-borne ligands for HUTS21 epitope exposure. Consistently, both FN (mean 350 μg/ml) and sVCAM-1 (mean 4.8 μg/ml) were detected in the plasma of CLL cases, irrespective to HUTS21 positivity. According to these observations, variable constitutive VLA-4 activation states were observed in CLL cells collected at the pre-treatment stage from patients undergoing IB treatment (VLA-4 RO ranging from 0.22 to 0.40); these values significantly decreased in CLL cells collected at day 14 (p=0.03) and day 30 (p=0.02) on IB, suggesting an IB-dependent impairment of antigen-independent (autonomous) BCR signals. The variability of the constitutive VLA-4 activation levels observed at pre-treatment was paralleled by variable VLA-4 activation levels upon BCR triggering (VLA-4 RO ranging from 0.56 to 0.65). Of note, an inverse correlation between the VLA-4 constitutive level and the extent of anti-IgM induced-VLA-4 activation was found (n=19, rho=-0.5, p=0.0093), implying a competition between antigen-independent and antigen-driven BCR signalling. Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a subset of CLL which might be connected to continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling, which is currently under evaluation. Disclosures Zaja: Takeda: Honoraria; Sandoz: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria; Abbvie: Honoraria.

Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

Background Chronic lymphocytic leukemia (CLL) is characterized by a CD20 expression lower than other lymphoproliferative disorders, hampering treatment efficacy of anti-CD20 antibodies. Although most mechanisms behind this phenomenon are still obscure, we demonstrated that mutations of NOTCH1 associate with a lower CD20 expression, ascribed to a NOTCH1 mutation-driven repression of CD20 transcription by hystone deacetylases (Pozzo et al. Leukemia 2016). This may explain the association between NOTCH1 mutations and clinical resistance to anti-CD20 immunotherapy in CLL patients treated with FCR (Stilgenbauer et al. Blood 2014). SF3B1 mutations affect ~10% CLL cases at diagnosis; a recent characterization of their functional meaning highlighted transcriptome-wide alterations of splicing patterns affecting several pathways, including the NOTCH1 pathway (Wang et al. Cancer Cell 2016). In this context, one of the most aberrantly-spliced genes is DVL2, a key component of the Wnt pathway and negative regulator of the NOTCH1 pathway. Aim To investigate CD20 expression in SF3B1-mutated CLL and the correlation with a putative secondary activation of the NOTCH1 pathway through splicing alterations of the NOTCH1 negative regulator DVL2. Methods Mutational status of NOTCH1 and SF3B1 was evaluated by next generation sequencing. Mutations with VAF-variant allele frequency below 2% were discarded. CD20 expression was evaluated by flow cytometry on the CD19+5+ population. Percentage of low-CD20 expressing (CD20dim) population was defined as %P1-(100-%P1), P1 being a linear gate spanning from zero to CD20 mode. Transcript levels of MS4A1 (encoding for CD20 protein) and spliced DVL2 were evaluated by QRT-PCR in cases with at least 75% of CD19+5+ cells. Statistical analyses were performed using Mann-Whitney rank-test. Results In a cohort of 537 unselected CLL cases, 93 cases (17.3%) carried NOTCH1 mutations: 62 delCT, 20 other truncating, 11 3′UTR (VAF range 3-84%, mean 32%; NOTCH1-mut); 46 cases carried SF3B1 mutations (8.5%, VAF range 5-53%, mean 32%; SF3B1-mut), the hotspot surrounding K700 being the most frequent (50%) followed by the hotspot surrounding G742 (30%); 7 cases (1,3%) carried both NOTCH1 and SF3B1 mutations. As trisomy 12 CLL is characterized by increased expression of CD20 and mutation of SF3B1 co-occurred in only 5/121 cases, the trisomy 12 subset was excluded from further analyses. Confirming previous findings, NOTCH1-mut cases were characterized by a lower CD20 mean fluorescence intensity (MFI) compared to WT cases (p=0.0154). In agreement with the hypothesis of a more active NOTCH1 pathway, CD20 expression was found diminished also in those cases carrying SF3B1 mutations, compared to WT cases (p=0.0059). Since CD20 expression is highly variable between cases, spanning a 2/3-log range of fluorescence intensity, we evaluated the percentage of the CD20dim population, this quantity being independent from the actual MFI. This analysis confirmed that CLL cases with NOTCH1 or SF3B1 mutations show a greater proportion of CD20dim cells (NOTCH1-mut p Presence of the alternatively spliced isoform of DVL2 (altDVL2) was identified only in those CLL cases with SF3B1 mutations (p35%) to isolate CD20high and CD20low populations. MS4A1 expression (higher in the CD20high subset) inversely correlated with altDVL2 expression (Figure 1c). As DVL2 can act as negative regulator of NOTCH1, we followed the hypothesis that loss of WT DVL2 due to alternative splicing may result in a more active NOTCH1 pathway. To simulate loss of DVL2 in the SF3B1-wild type CLL-like MEC1 cell line, cells were trasfected with siRNA for DVL2 itself and screened for CD20 expression. After 24 hours from transfection, CD20 expression was found downregulated at both protein (p=0.0062; Figure 1d) and transcript (p=0.0129) level. Conclusions In addition to NOTCH1-mut cases, also CLL cases bearing SF3B1 mutations are characterized by a lower CD20 expression, allegedly through a more active NOTCH1 pathway, potentially resulting in clinical resistance to anti-CD20 monoclonal antibodies. Disclosures Zaja: Amgen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; Sandoz: Honoraria; Abbvie: Honoraria.

CD49d, the alpha-chain of the integrin heterodimer α4β1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P

Background. NOTCH1 mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1 mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010). Aim. To investigate whether NOTCH1 mutations could affect CD20 expression in CLL. Methods. NOTCH1 mutations and NOTCH1 mutational burden were evaluated by ARMS PCR, QRT-PCR, conventional and next generation sequencing. NOTCH1 protein levels were evaluated by western blot. CD20 expression was evaluated by flow cytometry. Transcript levels of MS4A1, encoding for CD20 protein, were evaluated by QRT-PCR. CLL cells and CLL-like cell line MEC-1 cells were treated with the HDAC inhibitor valproic acid (VPA) at a concentration of 3mM. Susceptibility to rituximab was evaluated by complement dependent cytotoxicity (CDC) assay. MEC-1 cells were transfected with a vector containing a NOTCH1 intracellular domain (NICD) carrying either the 7544-7545delCT or a stop codon at the beginning of NICD sequence (c.5304G>A), as control. Results. i) In a cohort of 452 CLL, 54 cases carried NOTCH1 mutations. NOTCH1-mutated cases (NOTCH1-mut) with high mutational burden showed 3.0- fold higher transmembrane NOTCH1 and 2.1- fold higher NICD protein expression compared to NOTCH1 wild-type cases (NOTCH1-wt). NOTCH1-mut showed a lower Mean Fluorescent Intensity (MFI) of CD20 expression than NOTCH1-wt both in trisomy 12 (tris12, 18 NOTCH1-mut vs. 44 NOTCH1-wt, p Conclusions. CLL cases carrying NOTCH1 mutations are characterized by low CD20 expression levels that may confer resistance to anti-CD20 immunotherapy. The low CD20 expression, at least in part due to HDAC-dependent repression mechanism(s), can be reverted by HDAC inhibitor therapy. Disclosures No relevant conflicts of interest to declare.

Background. NOTCH1 mutations have been associated with an aggressive clinical course in chronic lymphocytic leukemia (CLL), and may predict anti-CD20 immunotherapy failure. Although about 80% of NOTCH1 mutations are 2-bp deletions (delCT) causing a sequence frameshift within the PEST domain, other mutations may occur involving either the PEST domain, i.e. frameshift mutations other than delCT (FS), missense (MS) and nonsense (NS) mutations, or, more recently described, the 3'UTR NOTCH1 sequence (UTR). Aim. To evaluate the clinical impact of the different types of NOTCH1 mutations in terms of time-to-first-treatment (TTFT) and overall survival (OS). Methods. NOTCH1 mutations were screened in 1003 CLL cases by NGS in exon 34 and part of 3'UTR with at least 1000X coverage and of 1% sensitivity. In selected cases, somatic mutations were confirmed by sequencing of germ line DNA from purified T cells. In our cohort, 529 patients were treated and 151 died. Cytogenetics distribution (n=970) was: 276, normal karyotype; 110, del11; 132, tris12; 369, del13; 83, del17. IGHV gene status (n=891) was: 529, (UM); 362, mutated (M). Results. i) Clinical impact of NOTCH1 mutations and correlation with cytogenetics and IGHV gene status. Collectively, NOTCH1 mutations were detected in 190 cases (18.9%): 167 cases bore a single mutation, while 19, 3 and 1 cases bore 2, 3 or 4 mutations, respectively. According to previous studies, cases with NOTCH1 mutations presented a significant shorter TTFT and OS respect to NOTCH1-wild-type (NOTCH1-wt) cases (p ii) Subclonal analysis of NOTCH1 mutations. Considering the threshold of variant allele frequency of 12% for clonal and subclonal definition, NOTCH1 mutated patients were divided into 118 clonal and 72 subclonal cases. Again in agreement with literature data: a) cases with subclonal NOTCH1 mutations experienced shorter TTFT than NOTCH1-wt cases (p=0.0184), but significantly longer than cases with clonal mutations (p iii) Clinical impact of different types of NOTCH1 mutations. The different types of NOTCH1 mutations (delCT, FS, MS, NS, UTR) were evaluated for their putative different prognostic impact in CLL. In order to assign a patient to a given NOTCH1 mutation type, cases presenting more than one mutations were classified using the mutation type presented at the higher percentage. Therefore, the NOTCH1 mutated cases were reclassified as follows: 123 delCT, 12 FS, 8 MS, 17 NS, and 30 UTR. According to Kaplan-Meyer curves, cases belonging to the five categories revealed a significant shorter TTFT respect to NOTCH1-wt cases (delCT p Conclusion. Our data confirm the capacity of NOTCH1 mutations to predict both TTFT and OS, although subclonal NOTCH1 mutations seem to have a prognostic relevance more for TTFT than OS. Moreover, we demonstrated for the first time that frameshift mutations different from the canonical delCT NOTCH1 mutation predicted for significantly shorter TTFT and OS compared to the other types of NOTCH1 mutations. A screening of NOTCH1 mutations in CLL, also outside the canonical region involved in delCT mutations, is therefore warranted for a correct identification of NOTCH1 mutated cases. Disclosures Rossi: Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau.

Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein. Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels. Methods. NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI). Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3'UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3'UTR NOTCH1 mutations (3'UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3'UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3'UTR group according to the substantially higher mutational burden detected for the 3'UTR mutation. ii)NOTCH1protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3'UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3'UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. Disclosures No relevant conflicts of interest to declare.

Background: stabilizing mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al, Blood, 2012; Del Poeta et al, Br J Haematol, 2013; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform. Aim: to identify molecular/biological features of NOTCH1 mutated CLL. Methods: the presence of NOTCH1 mutations was investigated by ARMS-PCR or by direct sequencing. The percentage of NOTCH1 mutated DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR) and/or by next-generation-sequencing. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4x44K platform. Specific gene/protein validations were performed by QRT-PCR, western blotting, flow cytometry and immunofluorescence. Cell proliferation was evaluated by Cell Trace Violet assay. CLL-like cell line MEC-1 was transfected with a vector either with a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or with a NICD carrying a missense mutation (c.5304G>A), generating a stop codon at the beginning of the sequence, as control. Results:i) in a cohort of 588 IGHV-UM CLL, 144/588 (24.5%) cases carried NOTCH1 mutations (NOTCH1-mut cases), with a percentage of NOTCH1 mutated DNA ranging from 1% to 37%. NOTCH1-mut cases (8 cases; 11%-37% of NOTCH1 mutated DNA) showed higher NOTCH1 protein expression than NOTCH1 wild type cases (NOTCH1-wt, 11 cases) employing monoclonal antibodies either recognizing the trans-membrane (mean fold-change=3.0) or the intracellular (mean fold-change=2.1) NOTCH1 domain. ii) A GEP comparing purified cells of 5 IGHV-UM NOTCH1-mut cases (15%-37% of NOTCH1 mutated DNA) and 5 IGHV-UM NOTCH1-wt cases selected nucleophosmin-1 (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-mut CLL. A higher expression of NPM1 and of the genes codifying for 4 ribosomal proteins in NOTCH1-mut cases was validated in a wider independent series of 34 cases by QRT-PCR (18 NOTCH1-mut cases).iii) Western blot in 19 cases (8 NOTCH1-mut cases) confirmed a higher NPM1 protein expression in NOTCH1-mut cases (1.3-5.2 range of fold-change) also at protein level. When intracellular distribution and fluorescent intensity of NPM1 immunostaining were evaluated, an additional cytoplasmic staining was visible in NOTCH1-mut cases, along with a clear nucleolar staining visible in both NOTCH1-mut and NOTCH1-wt cases. NPM1 protein expression was also determined by an intra-nuclear staining by flow cytometry. By using this approach, 2 NOTCH1-mut cases were sorted according to NPM1 expression; notably, the NPM1high subpopulation showed a higher NOTCH1 mutational load than the NPM1low subpopulation. iv) MEC-1 cells transfected with mutated NICD showed higher NOTCH1 transcript and protein levels than MEC-1 cells transfected with the control vector (increments over the control: NICD transcript =2.2, NICD protein =1.3). These NICD mutated MEC-1 cells were characterized by higher NPM1 transcript and protein levels than the MEC-1 cells transfected with the control vector (increments over the control: NPM1 transcript =2.0, NPM1 protein =1.5). By performing a cell-trace-violet proliferation assay, NICD mutated MEC-1 cells were characterized by higher proliferation rates than MEC-1 cells transfected with the control vector (p=0.018). Finally, when the transfected MEC-1 cells were treated with 0.5 microM etoposide for 48 hours, NICD mutated MEC-1 cells presented higher viability rates than the MEC-1 cells transfected with the control vector, as evaluated by Annexin V/7-AAD staining (percentage of viable cells: 70% vs. 50%). Conclusions: NPM1 was constitutively overexpressed in NOTCH1-mut IGHV-UM CLL together with several ribosome-associated components. An increased activity of the ribosomal machinery/DNA-repair mechanisms (Lindstrom MS. Biochem.Res.Int. 2011) in NOTCH1-mut CLL can confer proliferative advantages and resistance to chemotherapeutic agents. Disclosures No relevant conflicts of interest to declare.