B-CLL Cells Research Articles

Aberrant spliceosome activity via elevated intron retention and upregulation and phosphorylation of SF3B1 in chronic lymphocytic leukemia

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in CLL cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-Seq transcripts obtained from B cells (n=98 CLL patients) and healthy volunteers (n=09). We measured intron/exon ration to use that as a surrogate for ARS and found that 66% of CLL B-cells transcripts had significant IR elevation compared to NBC and that correlated with mRNA downregulation and low expression levels. Transcripts with highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 in CLL B-cells compared to NBC. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Targeting the NTSR2/TrkB oncogenic pathway in chronic lymphocytic leukemia

Current therapies that target the B-cell receptor pathway or the inhibition of anti-apoptotic proteins do not prevent the progressive forms of chronic lymphocytic leukemia (CLL), have low long-term efficacy and are subject to therapeutic resistance. Deciphering the mechanisms of leukemic cell survival and searching for new specific targets therefore remain major challenges to improve the management of this disease. It was evidenced that NTSR2 (neurotensin receptor 2), through the recruitment of TRKB (tropomyosin related kinase B), induces survival pathways in leukemic B cells. We have investigated the therapeutic potential of this protein complex as a new target. The binding domain of NTSR2 and TRKB was identified and a peptide targeting the latter was designed. The peptide binds TRKB and efficiently decreases the interaction of the two proteins. It is also effectively internalized by CLL-B cells in which it notably affects Src family kinase signaling and anti-apoptotic proteins levels. It demonstrated a cytotoxic effect both in vitro on the MEC-1 cell line and ex vivo on a cohort of 30 CLL patients. Altogether, these results underline the therapeutic potential of the NTSR2/TRKB protein complex as a target in CLL and open new perspectives for the development of targeted therapies.

Untargeted metabolomics identifies metabolic dysregulation of sphingolipids associated with aggressive chronic lymphocytic leukaemia and poor survival.

Metabolic dependencies of chronic lymphocytic leukaemia (CLL) cells may represent new personalized treatment approaches in patients harbouring unfavourable features. Here, we used untargeted metabolomics and lipidomics analyses to isolate metabolomic features associated with aggressive CLL and poor survival outcomes. We initially focused on profiles associated with overexpression of the adverse metabolic marker glycosyltransferase (UGT2B17) associated with poor survival and drug resistance. Leukaemic B-cell metabolomes indicated a significant perturbation in lipids, predominantly bio-active sphingolipids. Expression of numerous enzyme-encoding genes of sphingolipid biosynthesis pathways was significantly associated with shorter patient survival. Targeted metabolomics further exposed higher circulating levels of glucosylceramides (C16:0 GluCer) in CLL patients relative to healthy donors and an aggressive cancer biology. In multivariate analyses, C16:0 GluCer and sphinganine were independent prognostic markers and were inversely linked to treatment-free survival. These two sphingolipid species function as antagonistic mediators, with sphinganine being pro-apoptotic and GluCer being pro-proliferative, tested in leukemic B-CLL cell models. Blocking GluCer synthesis using ceramide glucosyltransferase inhibitors induced cell death and reduced the proliferative phenotype, which further sensitized a leukaemic B-cell model to the anti-leukaemics fludarabine and ibrutinib in vitro. Specific sphingolipids may serve as prognostic markers in CLL, and inhibiting enzymatic pathways involved in their biosynthesis has potential as a therapaeutic approach.

A Single Bout of Exercise Enhances the Efficacy of Rituximab Against Autologous Chronic Lymphocytic Leukaemia B Cells Ex Vivo By Transiently Increasing Natural Killer Cell Frequency in Blood, and Simultaneously Mobilises CD5 +CD19 +CD20 + B Cells into Blood

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20 + target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells - into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood. In this pilot study n = 20 treatment naïve patients with CLL (mean ± SD: age = 62 ± 10 years; height = 174.0 ± 7.5 cm; body mass = 83.3 ± 16.8 kg; body fat = 31.7 ± 9.8 %; blood leukocytes = 30.70 ± 22.21 ×10 9/L; anaerobic threshold = 14.1 ± 2.9 mL.kg -1.min -1) participated. Participants cycled at a moderate intensity (15% above their anaerobic threshold) for ~30-minutes, with blood samples collected pre-, post-, and 1-hour post-exercise. Given the importance of CD16 + NK cells in evoking ADCC, we enumerated NK cell subsets in blood samples collected pre, post-, and 1-hour post-exercise by flow cytometry. As expected, exercise induced a preferential increase of CD56 +CD16 + (+255%, p < 0.001) and mature, cytotoxic CD56 +CD57 +CD16 + NK cells (+322%, p < 0.001) pre- to post-exercise. Next, using immunomagnetic negative separation, NK cells and primary B-CLL cells were isolated from blood pre- and post-exercise and incubated together with heat inactivated foetal calf serum, with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab mediated cell lysis increased by +129% following exercise ( p < 0.001), with no change in antibody independent NK cell lysis of B-CLL cells - independent of rituximab - following exercise ( p = 0.25). Blocking CD16 on NK cells - vital for ADCC - blunted the effects of exercise on B-CLL cell lysis ( p = 0.84). In a subset of patients ( n = 9) we also explored the effects of autologous time-point matched plasma (instead of heat inactivated foetal calf serum) on rituximab mediated ADCC, which evoked a +92% increase in lysis pre- to post-exercise ( p = 0.038). Collectively, our results suggest that augmented efficacy of rituximab mediated ADCC was driven by an increase in CD16 + NK cells. We posited that the enhancement to rituximab mediated ADCC would have greater, clinically relevant implications if there was also a concomitant exercise-induced mobilisation of B-CLL cells expressing CD20 into blood from different body tissues. Flow cytometry revealed a +63% increase in CD5 +CD19 +CD20 + B-CLL cells ( p = 0.002) in blood after exercise. Further analyses revealed that CD5 +CD19 +CD20 + B-CLL cells with a phenotype consistent with recent egress from lymphoid tissue (CD5 brightCXCR4 dim; 70%, p = 0.004) and B-CLL cells with a propensity to migrate to peripheral tissues (CD5 dimCXCR4 bright; 67%, p = 0.002) were mobilised, with no change to overall CD20 surface antigen density ( p = 1.0) - determined by median fluorescence intensity. Furthermore, exercise evoked a +69% ( p = 0.022) increase in CD5 +CD19 +CD20 + B-CLL cells expressing CD49d, which is considered one of the strongest predictors of CLL prognosis. Taken together, these data demonstrate that exercise increased the frequency of CD20 + B-CLL cells with lymphoid origins and prognostic relevance into the blood, therefore rendering them susceptible to rituximab mediated ADCC. Our results show that individual bouts of moderate intensity aerobic exercise temporarily increased the number of cytotoxic CD16 + NK cells, and CD20 + B-CLL cells in blood. Additionally, our ex vivo investigations demonstrated enhanced rituximab mediated ADCC following exercise. Thus, exercise could be explored as a means of improving clinical responses in patients receiving rituximab, and/or other anti-CD20 monoclonal antibodies such as, Obinutuzumab.

Analysis of Single B Cells Reveals a Double Drive of Endocytosis and Transcriptional Downregulation Acting on the CLL B-Cell Receptor In Vivo

The cells of virtually all CLL cases, including those with unmutated (U) Ig gene heavy-chain variable regions ( IGHV) of poorer prognosis (U-CLL), exhibit variable degree of anergy. This is defined by (auto)antigen-mediated low surface immunoglobulin M (sIgM), but not sIgD, expression and signaling capacity, both reversible in vitro. In U-CLL there is also a tumor-related highly asymmetric use of IGHV1-69 ( 51p1 allele) (~30% of all U-CLL vs ~1% of the normal B-cell repertoire), claiming selection by (auto)antigen. While the effect of (auto)antigen on sIgM down-modulation by endocytosis is well known, less is known about the biosynthetic mechanisms regulating sIgM expression. We investigated the phenotypic, functional, and transcriptional consequences of sIgM engagement in bulk and single cells of IGHV1-69+ve U-CLL, compared to IGHV1-69-ve U-CLL and non-tumor IGHV1-69+ve B cells (identified by G6 monoclonal antibody F(ab)‘). The functional consequences were measured by intracellular calcium [iCa 2+] mobilization assay following anti-IgM F(ab‘)2 engagement. Phenotypic and functional analyses of bulk IGHV1-69+ve U-CLL populations (n=32) documented homogeneously lower sIgM, but not sIgD, levels and signaling capacity compared to IGHV1-69-ve U-CLL (n=49). Also, sIgM engagement mobilized iCa 2+ more slowly than IGHV1-69-ve U-CLL. However, following in vitro culture in ‘antigen-free’ medium for 48 hours, sIgM levels and signaling capacity/speed recovered more rapidly in IGHV1-69+ve than IGHV1-69-ve U-CLL, indicating a more profound and dynamic control of Ig expression in the IGHV1-69+ve CLL cells. Single cell t-SNE plots of IGHV1-69+ve U-CLL (3 patients) and normal B cells (2 donors) exposed ±anti-IgM F(ab')2 identified 2 distinct unstimulated or anti-IgM stimulated B cell clusters, in which IGHM transcripts could also be compared. We found that the basal (unstimulated) IGHM transcript levels were downmodulated in CLL cells compared to normal B cells. However, while IGHM transcript levels reduced dramatically following 2 hours anti-IgM engagement in the stimulated normal B cells, further downmodulation was not visible in the stimulated CLL cells, suggesting that (auto)antigen induced IGHM transcript downmodulation had likely already occurred in those CLL cells in vivo. Combined kinetics studies of the IGHM transcripts by qPCR and sIgM levels in flow cytometry revealed that IGHM transcripts recovered in the CLL cells within 48 hours of culture in vitro. The increase of IGHM transcripts anticipated sIgM protein recovery of expression on the CLL cells in vitro. Instead, in the normal B cells there was no transcript or sIgM protein increase. These data in IGHV1-69+ve cells provide additional fundamental evidence of chronic (auto)antigen drive in CLL and a novel mechanism of antigen-mediated transcriptional control of sIgM levels. The antiapoptotic machinery provides valuable time to the circulating leukemic cells to resynthesize sIgM glycoprotein following downmodulation by endocytosis. Therefore, there is a danger of interrupting sIgM signaling by therapeutic approaches with BCR inhibitors alone. A coordinated inhibition of BCR signaling and cell survival is required to prevent sIgM recovery in CLL. We thank Dr Roy Jefferis, University of Birmingham, UK, for kindly providing the G6 hybridoma. Stuart Lanham and Luis Del Rio Fernandez should be considered joint first authors.

A Non-Canonical Role for the Glycosyltransferase Enzyme UGT2B17 as a Novel Constituent of the B Cell Receptor Signalosome.

In chronic lymphocytic leukemia (CLL), an elevated glycosyltransferase UGT2B17 expression (UGT2B17HI) identifies a subgroup of patients with shorter survival and poor drug response. We uncovered a mechanism, possibly independent of its enzymatic function, characterized by an enhanced expression and signaling of the proximal effectors of the pro-survival B cell receptor (BCR) pathway and elevated Bruton tyrosine kinase (BTK) phosphorylation in B-CLL cells from UGT2B17HI patients. A prominent feature of B-CLL cells is the strong correlation of UGT2B17 expression with the adverse marker ZAP70 encoding a tyrosine kinase that promotes B-CLL cell survival. Their combined high expression levels in the treatment of naïve patients further defined a prognostic group with the highest risk of poor survival. In leukemic cells, UGT2B17 knockout and repression of ZAP70 reduced proliferation, suggesting that the function of UGT2B17 might involve ZAP70. Mechanistically, UGT2B17 interacted with several kinases of the BCR pathway, including ZAP70, SYK, and BTK, revealing a potential therapeutic vulnerability. The dual SYK and JAK/STAT6 inhibitor cerdulatinib most effectively compromised the proliferative advantage conferred by UGT2B17 compared to the selective BTK inhibitor ibrutinib. Findings point to an oncogenic role for UGT2B17 as a novel constituent of BCR signalosome also connected with microenvironmental signaling.

Abstract 2966: Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells

Abstract Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2966.

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles.

In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix.

Orelabrutinib, Rituximab, and High-Dose Methotrexate (HD-MTX) in Newly Diagnosed Primary Central Nervous System Lymphoma (PCNSL): A Retrospective Analysis on Efficacy, Safety, and Biomarker

Introduction: PCNSL is a rare malignant tumor arising in the brain, spinal cord, eyes, or leptomeninges. HD-MTX-based regimens remain the first option for newly diagnosed PCNSL because of high response rates as demonstrated by numerous studies (Qing et al. Cancer Manag Res 2021). Bruton's tyrosine kinase (BTK) is a crucial part of the B-cell receptor signaling pathway, and BTK inhibition has been proven effective in treating CLL and other B-cell lymphomas (Yoshitaka et al. Neuro-Oncology 2021). Orelabrutinib is a newly developed BTK inhibitor with high selectivity and a high concentration in the cerebrospinal fluid (Zhang et al, EHA 2022). Recent evidence has shown the efficacy and safety of orelabrutinib in PCNSL (Wu et al., Invest New Drugs 2022). Therefore, we conducted a retrospective study of clinical outcomes in newly diagnosed PCNSL patients (pts) treated with orelabrutinib combined with rituximab and HD-MTX (RMO) regimen in routine clinical care. Methods: This retrospective cohort included the pts (aged 18-80 years) with newly diagnosed PCNSL who received at least 2 cycles of RMO regimen (rituximab 375 mg/m2 iv day 1; MTX 3.5 g/m2 iv day1; orelabrutinib 150 mg qd po; 3 weeks per cycle) as the induction therapy in a real-world setting between May 2021 and June 2022. It was proposed to receive orelabrutinib monotherapy as maintenance after RMO therapy when deemed necessary. Efficacy was assessed by MRI/PET per the International PCNSL Collaborative Group (IPCG) criteria. The primary endpoint was overall response rate (ORR); secondary endpoints were CR rate, progression-free survival (PFS), overall survival (OS), and safety. Results: Thirty-Four pts with newly diagnosed PCNSL (20 male; 24 non-GCB subtypes) were included, with a median age of 58.5 years (range: 30-79). Four (11.8%) pts had an ECOG performance status of ≥3. All the pts (100%) achieved certain responses (Figure 1) with a complete response in 21 pts (61.8%). Eighteen (52.9%) pts completed 6 cycles of RMO as induction, with an ORR of 94.44% (17/18) and CR rate of 88.89% (16/18) at the 6th cycle assessment. As of the cut-off date (June 30, 2022), 8 pts received orelabrutinib maintenance therapy and had a CR status. Median PFS was not reached (95% CI, 7 months-not reached; Figure 2), with a 6-month PFS rate of 97.0% (95% CI, 91.3%-100.0%). Median OS was not reached either. In 22 pts who were available for gene alterations profiling, 18 pts (81.8%, 18/22) had an MYD88 mutation and achieved an ORR of 100% (CR, 55.6%). Four of these 18 pts were found to have a mutation in CD79B simultaneously. Plasma and CSF samples were collected from 5 patients at 2 hours after 15-day consecutive administration of orelabrutinib. The median CSF concentration of orelabrutinib was 21.6 ng/ml (range, 15.9-48.5), and the median CSF/plasma free ratio was 58.6% (range, 38.6%-82.5%). All these 5 pts achieved a response (3 CR; 2 PR). Six of 34 (17.65%) pts experienced adverse events (AEs). The most common AE was white blood cell count decreased (8.82%). Only 2 (5.88%) pts experienced 4 ≥grade 3 AEs, including white blood cell count decreased, anemia, platelet count decreased, and alanine aminotransferase increased. No treatment-related death occurred. Conclusion: This retrospective data suggested that orelabrutinib combined with RM had an encouraging anti-tumor activity in newly diagnosed PCNSL pts, with a toleratable safety profile. A prospective clinical trial is on the way to confirming this finding, and the results will be publicly disclosed after its completion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

New Triazacycloalkane Derivatives as Cytotoxic Agents for CLL Treatment: From Proof of Concept to the Targeting Biomolecule.

The 1,4,7-tris-(2-pyridinylmethyl)-1,4,7-triazacyclononane ligand (no3py) and its bifunctional analogue no3pyCOOK were synthesized to investigate their action toward zinc(II) depletion related to the apoptosis phenomenon in chronic lymphocytic leukemia (CLL) cells. no3py was used as the "free" ligand, while its "graftable" derivative was conjugated on a newly synthesized bifunctional sialoglycan, 6'-SL-NH2, selected to specifically bind CD22 biomarker expressed on the B-CLL cell surface. Both compounds were produced with good yields thanks to a Sonogashira coupling reaction and an orthoester function, respectively, for the chelator and the targeting moiety. The newly reported bioconjugate 6'-SL-no3py was then obtained through a peptidic coupling reaction. Biological in vitro studies of no3py and 6'-SL-no3py consisting of real-time detection of cell health (cytotoxicity and proliferation) and caspases 3/7 activation (crucial enzymes whose activation triggers cell death signaling pathways) have been investigated. First, Ramos, Daudi, and Raji B-cell lines, which present different sensitivity to zinc(II) content variation, were incubated with no3py and 6'-SL-no3py. Then, a videomicroscope allowed the real-time monitoring of the morphological changes leading to cell death from the detection of the cytotoxicity, the antiproliferative effect, and the caspasic activity. In terms of mechanism, the Zn2+ chelator cytotoxic effect of no3py has been evidenced by a culture medium ion supplementation study and by the decrease of intracellular fluorescence of Zn-specific fluorophore zinquin in the presence of no3py and 6'-SL-no3py chelators. Finally, flow cytometry analysis with classical Annexin V staining was conducted to detect no3py- and 6'-SL-no3py-induced apoptotic cell death in B-CLL cells. Time-course analysis, using the Incucyte Live-Cell Analysis System, demonstrated that no3py induced cell death in a time- and dose-dependent manner with variability across cell lines. 6'-SL-no3py exhibited the same dose-dependent trend as no3py, showing the efficiency of the targeting moiety. In both cases, the chelators depicted proliferation curves that were inversely correlated with kinetic death. Morphological changes specific to apoptosis and caspase 3/7 activation were observed for the three cell lines treated with no3py and 6'-SL-no3py, highlighting their role as apoptotic agents. A higher concentration of 6'-SL-no3py is needed to reach 50% of the B-CLL mortality, confirming a targeting of the chelator to the cell membrane. Overall, our results proved that the biological properties of the triazamacrocyclic chelator still remain even after addition of the targeting moiety. The free chelator as well as the bioconjugate constitute promising cytotoxic agents for CLL therapy through apoptosis induction.

Abstract 2855: Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults with CD19-targeting CAR T-cell therapy showing an excellent initial curative response. However, 10-20% of patients who receive anti-C19 CAR T-cell treatment experience relapse due to CD19 antigen loss. B-cell activating factor receptor (BAFF-R) that is expressed on the surface of normal and neoplastic B cells mediates the survival of mature peripheral B cells along with the B-cell receptor. Since B-CLL is characterized by apoptotic disruption and subsequent accumulation of mature B cells, BAFF-R is a logical, novel target for CAR T-cell therapy directed against B-CLL, especially if CD19 antigen loss occurs. In this study, we first manufactured BAFF-R CAR T-cells by infecting healthy donor Tnaïve/memory (Tn/mem) cells with BAFF-R CAR lentivirus before evaluating the antitumor activity of BAFF-R CAR T-cells on wild type MEC-1 (a B-CLL cell line), modified MEC-1, and primary cell lines. Antigen-specific cytotoxicity and activation of the BAFF-R CAR T-cells or control CD19 CAR T-cells were determined upon incubation with B-CLL cell lines and patient-derived primary B-CLL by measuring degranulation and IFN-γ release, respectively. BAFF-R CAR T-cells demonstrated substantial cytotoxicity against MEC-1, CD19-deficient MEC-1, and primary B-CLL cell lines; CD19 CAR T-cells showed no effect on CD19-deficient MEC-1 cells but did show effects with primary B-CLL and MEC-1 cells. Similarly, the release of IFN-γ was greater when BAFF-R CAR T-cells were co-cultured with CD19-deficient MEC-1 or primary B-CLL compared to CD19 CAR T-cells co-cultured with CD19-deficient MEC-1. In summary, BAFF-R CAR T-cells exert significant antitumor properties not only on patient-derived primary B-CLL cells but also on a CD19-deficient B-CLL cell line. We, therefore, assert BAFF-R-targeting CAR T-cells to be a novel therapeutic avenue for patients newly diagnosed with B-CLL and patients experiencing relapsed B-CLL after anti-CD19 therapy. Citation Format: Yan Luo, Kaihin Lui, Tommy T. To, Andrew liu, Farah Yassine, Mohamed A. Kharfan Dabaja, Martha E. Gadd, Hong Qin. Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2855.

Delineating the mechanistic pathway and therapeutic potential of Siglec-6 in Chronic Lymphocytic Leukemia

Abstract Sialic acid-binding immunoglobulin-like lectins (Siglec) regulate immune function via glycan recognition. Here, we show for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in chronic lymphocytic leukemia (CLL). Siglec-6+ primary B-CLL cells have significantly enhanced adhesion and migration towards sTn+ CLL− bone marrow stromal cells. A Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and B-CLL cells to the spleen and bone marrow in NSG mice. Mass spectrometry analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, WASP recruitment and enhanced actin polymerization, which were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6+ MEC1 cells and B-CLL cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells and normal donor B cells respectively, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane and is important for promoting actin polymerization at the leading cell edge. To study the therapeutic potential of Siglec-6, we generated a Siglec-6+ CLL mouse model (Siglec-6 x TCL1). Ex vivo experiments with a Siglec-6/CD3 targeted bispecific antibody showed specific killing of Siglec-6+ leukemic B cells. In summary, our studies have identified a novel role for Siglec-6 in promoting DOCK8 dependent CLL cell migration and opens up new therapeutic avenues to eliminate Siglec-6+ malignant B cells. Ongoing studies are focused on evaluating in vivo efficacy of Siglec-6/CD3 targeted bispecific antibody. Natarajan Muthusamy: NIH-R21 Grant and Pelotonia Idea grant Jessica Nunes: Pelotonia Graduate Fellowship

Exploring a peptide nucleic acid-based antisense approach for CD5 targeting in chronic lymphocytic leukemia.

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

B-cell antigen receptor expression and phosphatidylinositol 3-kinase signaling regulate genesis and maintenance of mouse chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a frequent lymphoproliferative disorder of B cells. Although inhibitors targeting signal proteins involved in B-cell antigen receptor (BCR) signaling constitute an important part of the current therapeutic protocols for CLL patients, the exact role of BCR signaling, as compared to genetic aberration, in the development and progression of CLL is controversial. In order to investigate whether BCR expression per se is pivotal for the development and maintenance of CLL B cells, we used the TCL1 mouse model. By ablating the BCR in CLL cells from TCL1 transgenic mice, we show that CLL cells cannot survive without BCR signaling and are lost within 8 weeks in diseased mice. Furthermore, we tested whether mutations augmenting B-cell signaling influence the course of CLL development and its severity. The phosphatidylinositol-3-kinase (PI3K) signaling pathway is an integral part of the BCR signaling machinery and its activity is indispensable for B-cell survival. It is negatively regulated by the lipid phosphatase PTEN, whose loss mimics PI3K pathway activation. Herein, we show that PTEN has a key regulatory function in the development of CLL, as deletion of the Pten gene resulted in greatly accelerated onset of the disease. By contrast, deletion of the gene TP53, which encodes the tumor suppressor p53 and is highly mutated in CLL, did not accelerate disease development, confirming that development of CLL was specifically triggered by augmented PI3K activity through loss of PTEN and suggesting that CLL driver consequences most likely affect BCR signaling. Moreover, we could show that in human CLL patient samples, 64% and 81% of CLL patients with a mutated and unmutated IgH VH, respectively, show downregulated PTEN protein expression in CLL B cells if compared to healthy donor B cells. Importantly, we found that B cells derived from CLL patients had higher expression levels of the miRNA-21 and miRNA-29, which suppresses PTEN translation, compared to healthy donors. The high levels of miRNA-29 might be induced by increased PAX5 expression of the B-CLL cells. We hypothesize that downregulation of PTEN by increased expression levels of miR-21, PAX5 and miR-29 could be a novel mechanism of CLL tumorigenesis that is not established yet. Together, our study demonstrates the pivotal role for BCR signaling in CLL development and deepens our understanding of the molecular mechanisms underlying the genesis of CLL and for the development of new treatment strategies.

BET Inhibition As a Targeted Epigenetic Approach to Reverse T Cell Dysfunction in Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the clonal expansion of mature CD19+/CD5+ lymphocytes in the peripheral blood and secondary lymphoid organs. The accumulation of B-CLL cells yields profound immune defects in the CLL tumor microenvironment (TME), promoting evasion of immune surveillance that contributes to tumor persistence and thus relapsed/refractory disease. The bromodomain and extra-terminal domain (BET) family of proteins are epigenetic readers that bind acetylated histone residues to regulate transcription of numerous genes involved in critical CLL protumor pathways. Of the BET family proteins, BRD4 is overexpressed in CLL and highly enriched at super-enhancers of genes that regulate CLL-TME interactions such as B cell receptor pathway components, chemokine/cytokine receptors, and immune checkpoint molecules. Pan BET inhibitors (BET-i), such as PLX51107 (Plexxikon Inc.) significantly improve survival in aggressive CLL murine models. Here we demonstrate that blocking BRD4 function with PLX51107 (PLX5) can alleviate the inherent immune defects observed in CLL, hence reducing B-CLL induced T cell dysfunction and allowing for robust B-CLL cell elimination. This therapeutic strategy may be vital in overcoming frequent drug resistance and/or bolstering the anti-tumor effect of current CLL therapies.Methods: Primary leukemic B cells were isolated from the peripheral blood of CLL patients and co-cultured with healthy donor T cells to evaluate the effect of PLX5 (0.1-0.5μM) on CLL-induced T cell immunosuppression ex vivo via an array of flow cytometry assays. T cell proliferation was assessed using CFSE after 96 h co-culture with α-CD3/α-CD28 stimulation. Effector cytokine production was evaluated after 48 h co-culture in the presence of PMA/ionomycin (final 6 h) and brefeldin A (final 5 h). Immune inhibitory molecule surface expression was measured following 48 h co-culture with α-CD3/α-CD28 stimulation. To further validate our ex vivo findings, the E μ-TCL1 adoptive transfer model was used. Once disease onset was confirmed in recipient WT B6 mice (>10% CD45+/CD19+/CD5+ peripheral blood lymphocytes), mice were randomized to receive either PLX5 (20 mg/kg) or vehicle (VEH) equivalent daily by oral gavage for 4 weeks. Following treatment, mouse spleens were processed to evaluate exhaustion marker expression, T cell proliferation (CellTrace™ Violet, 72 h a-CD3/α-CD28 stimulation ex-vivo), and T-cell effector function (ex-vivo mitogenic stimulation, 6 h).Results: T cell proliferation indices were reduced following ex vivo co-culture with primary B-CLL cells (mean ± SEM for T cells vs. co-culture, 2.0 ± 0.13 vs. 1.57 ± 0.05; P<0.01). This suppression was significantly alleviated in 0.5μM PLX5-treated co-cultures (1.84 ± 0.08; P<0.01). In a similar fashion, the percentage of polyfunctional TNF-α+/IFN-γ+ CD4+ T cells markedly increased in PLX5-treated co-cultures (VEH vs. 0.5μM PLX5, 10.0% ± 0.76% vs. 15.2% ± 0.92%; P<0.01). Notably, BET inhibition with PLX5 also bolstered T cell inflammatory function (%TNF-α+/IFN-γ+) in the absence of B-CLL cells (VEH vs. PLX5, 12.9% ± 1.0% vs. 15.3% ± 0.69%; P<0.05). Remarkably, the expression of numerous immune inhibitory molecules (e.g., PDL1, PD1, CTLA4, LAG3) was consistently reduced between 1.8- and 3-fold in PLX5-treated co-cultures (0.1μM). In the adoptive transfer E μ-TCL1 model, mice receiving PLX5 displayed reduced expansion of B-CLL cells and increased T cell infiltration in the spleen (Fig. 1A). Splenic CD4+ T cells from PLX5-treated mice had significantly greater proliferative capacity (Fig. 1B) and pro-inflammatory functionality (Fig. 1C). Finally, PLX5 treatment markedly reduced the surface expression of immune inhibitory molecules (e.g., PDL1, LAG3, VISTA) on CD4+ and CD8+ T cells in the spleen (Fig. 1D). Studies to evaluate the effects of PLX5 on malignant B-CLL and T cells within the bone marrow niche and soluble factors in the plasma are ongoing. Collectively, our data indicate that the novel BET-i, PLX5, exerts beneficial immunomodulatory effects on T cells within the CLL TME.Conclusion: Epigenetic-targeted therapies such as BET-i have the potential to alleviate CLL-induced T cell dysfunction while eliminating B-CLL cells and preventing tumor expansion. Future profiling studies are pending to further illuminate how BET proteins regulate immune function in CLL. [Display omitted] DisclosuresLunning: AstraZeneca: Consultancy; Legend: Consultancy; Acrotech: Consultancy; ADC Therapeutics: Consultancy; Kyowa Kirin: Consultancy; Myeloid Therapeutics: Consultancy; Beigene: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Verastem: Consultancy; Janssen: Consultancy; Daiichi-Sankyo: Consultancy; Morphosys: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Spectrum: Consultancy; Kite, a Gilead Company: Consultancy. Vose: Kite, a Gilead Company: Honoraria, Research Funding. Powell: Plexxikon Inc.: Current Employment.

Defining the Biochemical Role of Sialic Acid-Binding Immunoglobulin-like Lectin-6 in Adhesion and Migration in Chronic Lymphocytic Leukemia

Introduction: Sialic acid-binding immunoglobulin-like lectins (Siglec) are a group of lectins that regulate innate and adaptive immune function via glycan recognition. We and others have shown overexpression of Siglec-6, a member of Siglec family, on B cells from patients with chronic lymphocytic leukemia (CLL) compared to normal donor derived B cells. While placental expression of Siglec-6 has been shown to regulate invasion of trophoblast cells by binding to glycodelin, the biochemical role of Siglec-6 in CLL patients is not known. We describe here for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in CLL. Biochemical mechanisms of Siglec-6 mediated cell adhesion and migration through DOCK8 dependent activation of Cdc42 associated with actin polymerization in CLL cells are presented. Further, the physiological relevance of Siglec-6/ DOCK8 axis in CLL cell adhesion and migration is validated using primary CLL patient samples, and genetically engineered loss of function Siglec-6 and DOCK8 mutant MEC1 CLL cell line. These studies thus elucidate the biological role of Siglec-6 in malignant CLL B cells and demonstrate therapeutic opportunities targeting Siglec-6 in CLL.Methods: Flow cytometry was used to analyze surface expression of Siglec-6 and sTn in CLL patients and normal donors. Transwell migration assay was used to assess in-vitro migratory role of Siglec-6. Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. Cdc42 activation was evaluated using a commercial kit which uses specialized PAK1-PBD agarose beads to pull down GTP-bound Cdc42. To study the in-vivo migratory role of Siglec-6, MEC1 CLL cell line or primary CLL cells were blocked with an isotype antibody or Siglec-6 targeted antibody and injected into the tail vein of NSG immunocompromised mice. 24 hrs later, mice were euthanized and spleens and BM were processed followed by flow cytometry analysis to determine the number of human CD45+ cells that have migrated.Results: We confirmed Siglec-6 overexpression on B cells from CLL patients when compared to B cells from normal donors. Interestingly, we also found expression of sTn on bone marrow stromal cells (BMSCs) derived from CLL patients but not healthy donors. Compared to Siglec-6 + CLL cells, Siglec-6 - CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) media containing sTn or sTn + CLL-BMSCs in cell adhesion and trans-well migration assays. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6 + MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6 + MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, which are both downstream targets of DOCK8 involved in cell migration. Further, sTn also promoted actin polymerization, an effect that was compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6 + MEC1 cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane.Conclusions: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. Siglec-6 signals via DOCK8 to mediate sTn ligand dependent actin polymerization. We have also shown that sTn promotes Cdc42 activation and WASP protein recruitment which are both essential for actin polymerization. Moreover, all these effects were prevented by CRISPR/Cas9 mediated knock out of Siglec-6 or DOCK8 in MEC1 CLL cell line. Thus, Siglec-6 represents a CLL-specific target that opens up new therapeutic avenues to target only malignant B-CLL cells. Ongoing studies are focused on determining molecular mechanisms of Siglec-6 mediated regulation of actin polymerization and CLL-BMSC interactions.[This work was supported by NIH-R21 Grant and Pelotonia Idea grants. JN is a recipient of Pelotonia Graduate Fellowship] DisclosuresByrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria.

Identification of altered cell signaling pathways using proteomic profiling in stable and progressive chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by significant biologic and clinical heterogeneity. This study was designed to explore CLL B-cells' proteomic profile in order to identify biologic processes affected at an early stage and during disease evolution as stable or progressive. Purified B cells from 11 untreated CLL patients were tested at two time points by liquid chromatography-tandem mass spectrometry. Patients included in the study evolved to either progressive (n=6) or stable disease (n=5). First, at an early stage of the disease (Binet stage A), based on the relative abundance levels of 389 differentially expressed proteins (DEPs), samples were separated into stable and progressive clusters with the main differentiating factor being the RNA splicing pathway. Next, in order to test how the DEPs affect RNA splicing, a RNA-Seq study was conducted showing 4217 differentially spliced genes between the two clusters. Distinct longitudinal evolutions were observed with predominantly proteomic modifications in the stable CLL group and spliced genes in the progressive CLL group. Splicing events were shown to be six times more frequent in the progressive CLL group. The main aberrant biologic processes controlled by DEPs and spliced genes in the progressive group were cytoskeletal organization, Wnt/β-catenin signaling, and mitochondrial and inositol phosphate metabolism with a downstream impact on CLL B-cell survival and migration. This study suggests that proteomic profiles at the early stage of CLL can discriminate progressive from stable disease and that RNA splicing dysregulation underlies CLL evolution, which opens new perspectives in terms of biomarkers and therapy.

Pharmacodynamic Evaluation of Acalabrutinib in Relapsed/Refractory and Treatment-Naive Patients with Chronic Lymphocytic Leukemia (CLL) in the Phase 1/2 ACE-CL-001 Study

Pharmacodynamic Evaluation of Acalabrutinib in Relapsed/Refractory and Treatment-Naive Patients with Chronic Lymphocytic Leukemia (CLL) in the Phase 1/2 ACE-CL-001 Study

Dynamics of Gene Expression of Surface Receptors of Apoptosis in Primary Patients with B-CLL during Induction Treatment

Introduction. The study of the regulation of the expression of the genes of the external pathway of apoptosis in tumor cells in patients with B-CLL is an urgent task that allows optimizing therapy.Objective: To study the external pathway of apoptosis of tumor cells in B-CLL treated with RFC.Materials and methods. The study involved 10 patients with a newly diagnosed B-CLL 2-3 stage in Rai. Median age is 60 years. Patients received courses of PolychemotherapyRFC (rituximab + fludarabine + cyclophosphamide). The expression level of the main genes of the external pathway of apoptosis (DR3, DR4 / 5, FAS, TNFR2 and TRAIL) was determined by the RQ-PCR method. The parameter of the investigated parameters was determined before and after the course of PCT. Quantification of gene expression was performed with respect to the expression of the ABL gene taken as 100%. Statistical analysis of the results was carried out using STATISTICA10 software.Results. After the induction treatment of RFC, the average expression level of the DR3 gene increased from 553.5% to 1037.3% (p <0.05). Activity DR4 / 5 after therapy was characterized by a tendency to decrease from 727.01% to 543.9% (p <0.05). FAS activity decreased from 6855.9% to 848.1% (p <0.05). The expression level of TNFR2 increased from 1597.4% to 9618.3% (p <0.05), and the expression level of the TRAIL gene increased from 5801.3% to 6417.2% (p <0.05). Such dynamics of the surface receptor apoptosis gene indices was accompanied by clinical improvement in all patients, determined by the level of tumor lymphocytes (a decrease in the average values from 128 thousand to 26.73 thousand) and a 50% decrease in the size of peripheral lymph nodes.Conclusion. Thus, it is possible that DR3, TNRF2 and TRAIL are the most significant of the surface receptor apoptosis genes being studied, as the increase in their expression is correlated with the positive antitumor response. Reducing the level of expression of DR4 / 5 and FAS genes may indicate their lesser role in achieving a direct antitumor response. DisclosuresNo relevant conflicts of interest to declare.

Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm3 (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context. DisclosuresNo relevant conflicts of interest to declare.