Levels Of CD44 Research Articles

Inhibitory receptor CD47 binding to plasma TSP1 suppresses NK-cell IFN-γ production via activating the JAK/STAT3 pathway during HIV infection

BackgroundNatural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear.MethodsFresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling.ResultsCD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1–CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK–STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production.ConclusionsThe current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV.

The Expression Level of CD47 in Glioma and its Impact on Invasiveness

The Expression Level of CD47 in Glioma and its Impact on Invasiveness

Simplified Synthesis of Renieramycin T Derivatives to Target Cancer Stem Cells via β-Catenin Proteasomal Degradation in Human Lung Cancer.

Cancer stem cells (CSCs) found within cancer tissue play a pivotal role in its resistance to therapy and its potential to metastasize, contributing to elevated mortality rates among patients. Significant strides in understanding the molecular foundations of CSCs have led to preclinical investigations and clinical trials focused on CSC regulator β-catenin signaling targeted interventions in malignancies. As part of the ongoing advancements in marine-organism-derived compound development, it was observed that among the six analogs of Renieramycin T (RT), a potential lead alkaloid from the blue sponge Xestospongia sp., the compound DH_32, displayed the most robust anti-cancer activity in lung cancer A549, H23, and H292 cells. In various lung cancer cell lines, DH_32 exhibited the highest efficacy, with IC50 values of 4.06 ± 0.24 μM, 2.07 ± 0.11 μM, and 1.46 ± 0.06 μM in A549, H23, and H292 cells, respectively. In contrast, parental RT compounds had IC50 values of 5.76 ± 0.23 μM, 2.93 ± 0.07 μM, and 1.52 ± 0.05 μM in the same order. Furthermore, at a dosage of 25 nM, DH_32 showed a stronger ability to inhibit colony formation compared to the lead compound, RT. DH_32 was capable of inducing apoptosis in lung cancer cells, as demonstrated by increased PARP cleavage and reduced levels of the proapoptotic protein Bcl2. Our discovery confirms that DH_32 treatment of lung cancer cells led to a reduced level of CD133, which is associated with the suppression of stem-cell-related transcription factors like OCT4. Moreover, DH_32 significantly suppressed the ability of tumor spheroids to form compared to the original RT compound. Additionally, DH_32 inhibited CSCs by promoting the degradation of β-catenin through ubiquitin-proteasomal pathways. In computational molecular docking, a high-affinity interaction was observed between DH_32 (grid score = -35.559 kcal/mol) and β-catenin, indicating a stronger binding interaction compared to the reference compound R9Q (grid score = -29.044 kcal/mol). In summary, DH_32, a newly developed derivative of the right-half analog of RT, effectively inhibited the initiation of lung cancer spheroids and the self-renewal of lung cancer cells through the upstream process of β-catenin ubiquitin-proteasomal degradation.

Tafasitamab in Combination with a CD20xCD3 Bispecific T-Cell Engager Significantly Prolongs Survival in Preclinical Lymphoma Models

R-CHOP remains the standard of care for newly diagnosed DLBCL patients. However, 30-40% of patients are refractory or relapse after initial response to the immunochemotherapy, indicating a high-unmet medical need for these patients. Tafasitamab is a CD19-targeting, Fc-modified humanized monoclonal antibody mediating antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity. Tafasitamab was granted accelerated approval by the FDA in 2020 and conditional marketing authorization by the EMA in 2021 for the treatment of transplant-ineligible adult patients with relapsed or refractory (R/R) DLBCL in combination with the immunomodulatory drug lenalidomide. CD20xCD3 T-cell engagers are currently under clinical investigation or recently received FDA approval in 2L+ DLBCL (Thieblemont 2023; Dickinson 2022). CD19- and CD20-targeting antibodies might complement each other, as I) CD19 could compensate for CD20-low/ -negative settings and vice versa and II) monoclonal antibodies and T-cell engagers combine powerful and distinct immune pathways holding great potential to increase responses in patients who may need additional therapeutic options. The aim of this study was to investigate the combination of tafasitamab and MOR212, a proprietary proof-of-concept CD20xCD3 T-cell engaging antibody, in preclinical models of lymphoma. First, MOR212 was characterized in vitro and in vivo to ensure comparability with other CD20xCD3 bispecific antibodies (comp1-3) currently under clinical investigation. In in vitro cytotoxicity assays, MOR212 demonstrated similar (comp1-2), or improved (comp3) cytotoxicity (MOR212 vs comp1 and 2 p>0.05 vs comp3 p=0.03; exemplary data for EC50 in SU-DHL-4 cells; n=6), cytokine secretion (MOR212 vs comp1-3 p>0.05; exemplary data for EC50 TNFα; n=4) and T-cell activation (MOR212 vs comp1 p=0.05; vs comp2 p>0.05 vs comp3 p=0.003; exemplary data for % CD69 on CD8 T-cells; n=6) in cell lines expressing different levels of CD20. In a lymphoma in vivo model co-engrafted with human T-cells, MOR212 prolonged progression-free survival of mice to a similar extent as two comparator molecules that were tested head-to-head (MOR212 vs comp1 and 3 p>0.05;) at a dose of 0.1 mg/kg. Next, we investigated the combination of tafasitamab and MOR212 in vitro. Due to the differential killing kinetics of a monoclonal antibody and a T-cell engager, we made use of a real time live-cell imaging assay. Lymphoma cells were co-cultured with healthy donor peripheral blood mononuclear cells (PBMCs) and tafasitamab and MOR212 were added in different concentration combinations. Tumor cell killing was monitored every 3 hours for 72 hours and revealed improved target cell killing by the combination of both compounds (signal/background caspase 3/7 staining at 48h: MOR212 24.2 vs tafasitamab 28.2 vs MOR212+tafasitamab 111.5; n=1). To confirm these findings in vivo, we used human CD34+ engrafted NCG mice with a huIL15 boost allowing for the development of human T-cells as well as NK-cells, as essential effector cells for the combination of both compounds. Raji lymphoma cells were engrafted and treatment with the single or combination therapy was started once tumors reached a pulpable size. Combination of tafasitamab and MOR212 significantly improved progression-free survival in animals compared to monotherapy treatment (tafasitamab+MOR212 vs tafasitamab only p=0.0002 and MOR212 only p=0.0098). In addition, the combined treatment regimen appeared to be safe in mice as no side effects on both body weight loss and clinical score were recorded throughout the entire observation period. Our findings prove that the combination of tafasitamab and a CD20xCD3 T-cell engaging antibody significantly increases tumor cell eradication both in vitro and in vivo and lay the ground to potentially translate the results into improved outcome for patients with aggressive lymphoma in future clinical trials.

Analysis of Tagraxofusp Activity in AML-Pdc As a Single Agent and in Combination with BCL2 Inhibitors

Acute myeloid leukemia with increased plasmacytoid dendritic cells (AML-PDC) is a new leukemia recently clarified in the classification of hematological diseases (WHO 2022). AML-PDC presents with leukemic CD34+ blasts associated with an excess of pDCs, both of which are characterized by the same mutational profile. Xiao et al. (Blood 2021) also demonstrated that certain leukemic blasts upregulate a pDC transcriptional program, enabling these cells to differentiate into pDCs. Blasts and pDCs express the alpha-subunit of the interleukin-3 receptor, known as CD123, at different levels. Tagraxofusp, a first-in-class CD123-directed therapy, is a recombinant fusion protein consisting of human interleukin-3 conjugated to a truncated diphtheria toxin payload. Tagraxofusp is approved for the treatment of BPDCN (US/EU), a disease where pDCs are critical factors in disease development. Given the elevated number of pDCs expressing high levels of CD123 and the lack of therapies available to treat AML-PDC, we evaluated tagraxofusp activity on AML-PDC models. We first evaluated tagraxofusp on primary cells from AML-PDC patients and characterized cytotoxicity by flow cytometry (FC) on both pDCs and blasts. We observed a decrease of pDC viability both in a time and concentration dependent manner, with viability reaching 32.8% ± 33.4% after 48 h of treatment at 2.33 µg/mL. We next evaluated sensitivity at different levels of IL-3R expression. We observed a significantly lower level of CD123 expression from AML-PDC patient blasts by FC than pDCs. This was similarly observed for CD131 expression, with a RFI of 2.2 ± 1.2 on blasts compared to 7.3 ± 4.7 on pDCs. The CD131 result was confirmed at the genomic level with scRNAseq analysis on two patients with a significantly lower expression of CSF2RB (coding for CD131) in the blasts compared to pDCs. Dimerization of CD123 and CD131 upon IL-3 binding activates downstream signaling pathways (such as the JAK/STAT pathway) and internalization of tagraxofusp. Given this and our findings related to CD131, we next evaluated phosphorylation of STAT5 as an IL-3 binding surrogate to IL-3R. We observed significantly lower STAT5 phosphorylation on blasts compared to the pDCs after 30 minutes of stimulation with tagraxofusp. Overall, blasts present a lower CD123 and CD131 expression compared to pDCs as well as a lower STAT5 phosphorylation after stimulation by tagraxofusp. Of note, pDCs from two patients presenting a low CD131 RFI also exhibited a lower sensitivity to tagraxofusp compared to pDCs from other patients. The low expression of these IL-3R components could explain the low sensitivity of blasts to tagraxofusp compared to pDCs in some patient samples. We further combined tagraxofusp with other available therapies used in AML (ie, BCL-2 inhibitors, proteasome inhibitors, or hypomethylating agents) to determine the optimum combination needed to eliminate both pDCs and blasts. A significant reduction in blast viability was observed when tagraxofusp was combined with venetoclax. Combination of tagraxofusp with venetoclax showed an increased reduction in blast viability compared to tagraxofusp alone, with a viability of 52.5% ± 13.1% and 82.6% ± 18.4%, respectively. Also, scRNAseq analysis showed a higher expression level of BCL-2, the target of venetoclax, in blasts compared to the pDCs; this could explain the differential venetoclax sensitivity between blasts and pDCs in AML-PDC. The treatment of AML-PDC is challenging, and the presence of CD123 over expressing leukemic blasts associated with excess pDCs, makes tagraxofusp an attractive treatment option in this setting. Tagraxofusp was able to effectively eliminate pDCs and blasts to a lesser extent as a single agent. High expression of BCL-2 in blasts supports the consideration of tagraxofusp in combination with venetoclax as an effective combination therapy to eradicate both blasts and pDCs in AML-PDC patient samples.

Higher CD56 Expression on Multiple Myeloma Cells Increases CD38 Expression, Reduces Intracellular NAD+ Levels, and Enhances the Efficacy of Daratumumab-Based Treatment Strategies

NCAM1 (Neural Cell Adhesion Molecule 1), also known as CD56, is a member of the immunoglobulin superfamily and is deregulated in many tumors. In multiple myeloma (MM), high levels of CD56 are observed on MM cells in 70% of patients. This molecule has several functions including tumor growth, adhesion and response to therapy, as suggested by several studies. However, an extensive analysis of the specific landscape underlying its presence and its role as a prognostic biomarker is lacking, especially in MM. Here, we evaluated CD56 surface levels on tumor cells collected from 136 newly diagnosed MM patients (Policlinic San Martino Hospital, Genoa, Italy) and found that low CD56 expression significantly correlated with extramedullary disease (EMD) (p = .016) while high CD56 levels were associated with a lower risk of disease progression or death (p = .003). When evaluating the surface expression of MM cells, we surprisingly found a positive correlation between CD56 and CD38 levels (p < .0001), which was replicated in a panel of MM cell lines (p = .02). To gain insight into the biological significance of this, we challenged a panel of MM cell lines with Daratumumab (DARA), a standard-of-care CD38 antibody that kills MM cells through multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC). Given the correlation between the expression of CD56 and CD38, we hypothesized that higher CD56 expression would lead to enhanced DARA-mediated ADCC. We performed standard 3-hour ADCC assays using NK cells from healthy donors as effector cells and CD56-positive or -negative cell lines. Myeloma cells were stained with calcein-AM and co-cultured with NK cells at indicated effector:target (E:T) ratios. The assay was assessed in the presence or absence of 1μg/mL Daratumumab. We found that MM cell lines with high expression of CD56 were more sensitive to DARA-mediated NK cell killing (p = .004 at ratio 1:1 E:T). Consistently, MM patients with higher levels of CD56 (N = 8) had better overall response rates to DARA-based therapies compared to patients with lower levels (N = 9). CD56 and CD38 are not known to interact, so this correlation may be mediated by the NAD+ biosynthesis pathway. CD38 is a critical enzyme for breaking down NAD + in cells. We found that ectopic expression of CD56 led to lower intracellular NAD + levels (MM1S overexpressing CD56 p = .0875; RPMI 8226 overexpressing CD56 p = .0003) and increased CD38 enzymatic activity (MM1S overexpressing CD56 p = .0035; RPMI 8226 overexpressing CD56 p = .0478), as measured by a cyclase activity assay. As a result, CD56 surface levels affected MM cells' sensitivity to NAD +-lowering agents, including next-generation NAMPT inhibitors. Indeed, MTS assays confirm that CD56-overexpressing MM cells were more vulnerable to these small molecules, whereas CD56 silencing made cells more resistant. In conclusion, our data suggest CD56 levels as an important determinant of sensitivity to DARA-based therapies. Moreover, our results suggest the next-generation NAMPT inhibitors as an additional therapeutic strategy for CD56-expressing MM patients.

Preclinical Studies Demonstrating Efficacy of Tasquinimod in Models of Advanced Myeloproliferative Neoplasm (MPN) in Blastic Phase

The alarmins, S100A8 (A8) and S100A9 (A9), are low molecular weight proteins belonging to the S100 protein family. Also called calprotectins, A8 and A9 can form homodimers or heterodimers and bind Ca++. A8 and A9 are secreted into the extracellular space and plasma, where they interact with TLR4 (Toll like receptor 4), RAGE (Receptor for Advanced Glycation End products) and CD33. Intracellularly, they act as Ca++ biosensors and activate NADPH oxidase, stimulating ROS production, which activates NLRP3 inflammasome. A8 and A9 also amplify the inflammatory response through TLR4, and induce inflammatory cytokines, including TNFα and IL-6, through activation of NFkB and MAP kinase pathways. A8 and A9 are highly expressed in AML, especially myelomonocytic and monocytic AML, and are associated with poor prognosis. Through scRNA-Seq analysis, A8/A9 heterocomplex was shown to be highly expressed in myeloid and stromal cells and marked progression of bone marrow fibrosis in PMF (Primary Myelofibrosis). In the murine JAK2-V617F-driven PMF model, Tasquinimod (TM, Active Biotech), an orally active, quinoline-3-carboxamide linomide analogue immune-modulatory agent was shown to bind A9, inhibiting its interaction with TLR and RAGE receptors. Notably, TM treatment reduced myeloproliferation, splenomegaly and MF in JAK2-V617F mice. Therefore, based on these findings, in present studies, we determined in vitro and in vivo efficacy of TM against cellular models of advanced MPN cell lines and patient-derived (PD) CD34+ blastic phase (BP, > 5% blasts in PB) MPN cells. Treatment with TM (10 to 30 µM for 96 hours) induced loss of viability in the MPN-AML SET2, HEL92.1.7 cell lines and PD MPN-BP cells, but not in normal CD34+ progenitor cells. RNA-Seq analysis showed that exposure to TM (20 µM for 16 hours) led to negative enrichment of the gene-sets of MYC and E2F targets, of mTORC1 and IL6-JAK-STAT3 signaling, and of inflammatory response in PD MPN-AML cells. This was associated with log2 fold-decline in MYC, BCL2, CDK6, MPL, CCND1 and myeloperoxidase (MPO), but upregulation of mRNA of GFI1 and p21. CyTOF analysis revealed that TM treatment significantly reduced expressions of A8, A9, p-ERK, MPO, CXCR4, Cyclin D1, PU.1 and Ki67, but increased protein levels of GFI1, p21 and cleaved PARP in phenotypically defined PD, CD34+ MPN-BP cells expressing high levels of CLEC12A, CD99, CD123 but low expression of CD11b. Additionally, our findings showed that co-treatment with TM (5 to 30 µM) and ruxolitinib (250 to 1000 nM), BET inhibitor OTX015 (50 to 250 nM) or pelabresib (CPI-0610) (100 to 500 nM), or BCL2/Bcl-xL inhibitor navitoclax induced synergistic lethality in advanced MPN-BP cells exhibiting delta synergy scores of >1.0 (by the ZIP method). Notably, our findings also demonstrated that treatment with TM (10 or 30 mg/kg/day, by oral gavage) for 8-weeks, significantly improved survival without inducing any toxicity in immune-depleted NSG mice engrafted with PDX cells of MPN-AML with mutations in CALR and TERT. In a separate experiment on the same PDX model, treatment with TM (30 mg/kg/day) also induced significantly greater survival advantage than treatment with ruxolitinib (30 mg/kg/day) or OTX015 (30 mg/kg/day) by oral gavage. These findings clearly demonstrate preclinical efficacy of TM in advanced MPN-BP cellular models and create the rationale to further interrogate the efficacy of TM alone and in combinations with current, front-line therapies for advanced MPN with excess blasts.

RRMM and Post-BCMA Treated Subjects from the CC-220-MM-001 Study Show Increased Genomic Aberrations Associated with High-Risk and Significant Dysfunction in CD4+ T-Cell Compartment Compared to NDMM Subjects

Introduction Outcomes for patients with multiple myeloma (MM) have improved significantly through development of novel therapeutics, including immunomodulatory agents (IMiDs ®), proteasome inhibitors, anti-CD38 monoclonal antibodies and new BMCA targeting T-cell redirecting therapies. While most MM patients respond well to initial therapy, over time their disease relapses, requiring several subsequent lines of treatment. However, how patient tumors and immune profiles evolve over lines of treatment and how this may impact the efficacy of subsequent treatment regimens remains poorly understood. CC-220-MM-001 is a multicohort, phase 1/2 trial (NCT02773030) studying the efficacy of iberdomide (CC-220; a cereblon [CRBN] E3 ligase modulator) as monotherapy, and in combination with other treatments in subjects with newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM), including patients who have received prior BCMA targeting agents. This study allows uniform comparison of immune and tumor biomarkers across these clinical patient segments. Methods Baseline samples collected in the CC-220-MM-001 study prior to iberdomide treatment included peripheral blood for immunophenotyping, bone marrow biopsies for immunohistochemistry and CD138+ enriched bone marrow aspirates for genomic analysis. Biomarkers were evaluated for differences between NDMM subjects and subjects refractory to IMiD agents, proteasome inhibitors, anti-CD38 antibodies, and BCMA targeting CAR-T and T-cell engager therapies. Results Analysis of immune profiles showed NDMM subjects had a less immunodeficient profile compared to RRMM subjects, particularly in the CD4+ compartment, with significantly more total and naïve CD4+ T-cells (p<0.001) and significantly lower proportion of PD-1+ CD4+ T-cells (p=0.002) and CD4+ Tregs (p=0.005). RRMM subjects had median levels of several immune cells well below the lower limit of normal (LLN) reference range for healthy individuals, including low B cells, NK-cells, and total CD3+ and CD4+ (but not CD8+) T-cells. Median number of CD4+ T-cells was approximately half of the LLN (500 cells/mL) and median ratio of CD4+ to CD8+ T-cells was ~0.75. Phenotypic assessment of immune cells in RRMM subjects showed evidence of immune stimulation with >30% T-cells expressing activation markers (HLA-DR+ and ICOS+) and exhibiting a memory phenotype (CD45RO+). In contrast, a low proportion of pre-treatment T-cells were observed to be in a proliferative state (Ki-67+; median 6.2% of CD3+) despite evidence of activation, and more than 1/3 of both CD4+ and CD8+ cells in RRMM subjects expressed PD-1, together suggesting T-cell dysfunction and/or exhaustion. These results were mostly consistent regardless of prior refractoriness to IMiDs and PIs. Conversely, subjects post anti-CD38 had lower levels of NK and CD38+ T-cells (p<0.001) and subjects post-BCMA T-cell redirecting therapies (n=22) trended to have lower absolute levels of both CD4+ and CD8+ T-cells and increased proportion of T-cells expressing activation markers than other RRMM patient segments. Analysis of the bone marrow compartment showed that while CRBN protein was quantifiably detectable in CD138+ cells in almost all samples, including in subjects coming off IMiD based regimens, 40% of RRMM subjects had evidence of molecular dysregulation of CRBN including, loss of COPS7b/COPS8, high CRBN-del-exon10 expression, CRBN LOH or CRBN mutation. A wide range of CRBN protein levels was observed, however, differences in protein levels did not correlate with refractory status to any therapy. Notably, median CRBN expression levels were 2× higher in NDMM subjects. Prevalence of translocations, hyperdiploidy, CNAs and other mutations in RRMM subjects showed 31% of subjects had KRAS mutations, 28% had Amp1q, 23% were hyperdiploid, 21% had del17p, 12% had t(11;14), 11% had NRAS mutations and 9% had t(4;14). Conclusions These data illustrate that late-line RRMM subjects have appreciable immunosuppression compared to NDMM subjects, with particular dysfunction in the CD4+ helper T-cell compartment, suggesting that the efficacy of immunotherapies in late line myeloma may benefit from combinations with agents that improve CD4+ T-cell function. These data also show enrichment of molecular high-risk segments and CRBN-related genomic aberrations in RRMM subjects in the CC-220-MM-001 study.

Natural IgM Recognizing Oxidation-Specific Epitopes Attenuate NETosis Induced By Extracellular Vesicles

BACKGROUND Neutrophil extracellular traps (NETs) emerged as important contributors to thrombus formation. We have previously demonstrated the role of NETs in acute myocardial infarction (AMI). However, the mechanistic understanding of modulators of NETosis in the context of AMI is scarce. Levels of extracellular vesicles (EV) carrying lipid peroxidation-derived epitopes also called oxidation-specific epitopes (OSE), are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE have been shown to inhibit the pro-coagulatory and pro-inflammatory effects of EV, and low plasma levels predict cardiovascular risk. We hypothesized that large EV, also called microvesicles (MV), induce NETosis during AMI, and that natural anti-OSE IgM are inhibiting this process. METHODS Patients were consented when the decision for primary percutaneous intervention (pPCI) for acute ST-segment elevation myocardial infarction (STEMI) was made, and blood was aspirated from the culprit site and peripheral site as an in-patient systemic control (n=28). Myocardial function was assessed by cardiac magnetic resonance imaging (cMRI) 4±2 days and 195±15 days after pPCI. EV were isolated from patient culprit site plasma and cell culture supernatants and characterized by flow cytometry. Isolated EV were used for neutrophil stimulation in vitro, and in vivo using a murine injection model in the presence of the IgM LR04 recognizing the immunodominant OSE, malondialdehyde (MDA) epitopes, or an isotype control. NETs were visualized by immunofluorescence staining for DNA-histone, citrullinated histone 3 (citH3) and myeloperoxidase (MPO). NET markers and natural IgM recognizing OSE in murine and patient plasma were measured by ELISA. RESULTS Culprit site plasma contained more and proportionally higher levels of leukocyte-derived CD45+ MDA-EV compared to the peripheral site plasma. NET markers were associated with CD45+ MDA-EV at the site of occlusion. Decreased levels of MDA-specific IgM at the culprit site indicated consumption of protective IgM. EV isolated from AMI patient plasma induced NETosis in vitro and after injection into C57BL/6 mice in vivo, as determined by NET markers and fluorescence microscopy of histone citrullination in neutrophils. The malondialdehyde-specific IgM LR04, but not a control IgM, reduced the NETogenic effects of EV in vitro and in vivo. Higher circulating levels of EV and lower OSE-IgM were associated with reduced ejection fraction in AMI patients at follow-up. CONCLUSION EV from AMI patients induced NET formation in vitro and in vivo, and natural IgM recognizing malondialdehyde-epitopes attenuated this effect. In summary, the balance between OSE-EV and OSE-IgM at the culprit site during AMI may represent a potential prognostic and therapeutic target. Moreover, the modulatory effect of natural IgM may also apply to other pathologies where NETosis contributes to thrombus formation.

PBMC-Humanized Mouse Model to Assess Individual Variability in Efficacy and Toxicity of Universal CD19 Chimeric Antigen Receptor T Cell Immunotherapy

Chimeric antigen receptor T cell (CART) immunotherapy has been accumulating extraordinary breakthroughs in fighting against hematological malignancies. Particularly, universal, “off-the-shelf”, CARTs (uCARTs) are emerging as an attractive alternative to patient-derived CARTs, offering cost-effectiveness, immediate availability for treatment, and independence from patients' lymphopenic conditions. However, uCARTs share the same safety concerns with autologous CARTs while the efficacy and persistence of uCARTs vary by each product and remain uncertain. Further, T cells sourced from healthy donors for uCARTs may manifest individual-specific immune characteristics upon interaction with cancer cells and the patient's immune system. These characteristics often remain latent when assessed in vitro and complicate the prediction of uCART therapy's toxicity and efficacy on an individual basis. We previously demonstrated that the PBMC-humanized mouse model can capture efficacy and toxicity of autologous and allogeneic CD19 CARTs. We showed that the CART-induced toxicity varied by the combination of PBMC donors and CART cell donors. Here, we extend the use of PBMC-humanized mice to assess the potential toxicity, efficacy, and persistence of uCARTs. Female NSG™-DKO mice (n = 19-20 mice per donor) were irradiated and reconstituted with human PBMCs sourced from four selected donors (Donor A, B, C, and D) out of 58 available in our PBMC bank. The selection criteria were based on their pre-characterized in vivo human cytokine induction profiles, specifically IFN-gamma response levels to OKT3 treatment (Donor A: 268 pg/ml; B: 1936 pg/ml; C: 4862 pg/ml; D: 13538 pg/ml). PBMC-humanized mice were grouped into three different CD19 CART treatments; a) Allogeneic CARTs were manufactured from a healthy donor's T cells containing CD28 costimulatory domain (19-28z) and no genetic modification was made for “off-the-shelf” availability; b) uCART 1 cells (19-28z) were knocked out of TCRab via CRISPR-cas9 (19-28z); c) uCART 2 cells were knocked out of CD3 and TCRab using TALENs and magnetically depleted for remaining CD3/TCRab, and contain 4-1BB costimulatory domain. Body weight change and clinical scores were measured throughout the experiments. We measured the levels of human CD45, human CD19, and CART expansion from blood collected 8 days post-treatment using flow cytometry. We measured 54 human cytokine levels from serum collected 48 hours post-treatment using a multiplex assay. CART-induced toxicity, measured by body weight loss, dramatically varied by PBMC donors and the treatments (Figure 1). uCART 2 induced significant body weight loss in Donor B and D reaching the lowest 6 days post-treatment. Notably, all mice humanized with Donor C and treated with uCART 2 reached a humane endpoint within 12 days post-treatment. We only observed noticeable body weight loss from uCART 1 in Donor C. Allogeneic CART did not induce body weight loss across the four donors. Efficacy, measured by the % of elimination of human CD19 cells in peripheral blood compared to the PBS-treated group, also varied by PBMC donor and the treatments (Figure 2). Overall, Allogeneic CART showed the best elimination of CD19 cells. Mice humanized with Donor D responded with the poorest efficacy of the three CART treatments. CART expansion was observed only in uCART 2 treated mice that reached a humane endpoint within 6 days post-treatment. CART persistence measured 16 days post-treatment was not observed across three CARTs and this corresponds to the clinical data of uCARTs. Principal component analysis of human cytokine data revealed that uCART 2 induced the most distinct cytokine responses compared to the other CARTs, driven by IFN-gamma, IL-10, IL-5, IL-3, and GM-CSF. In Donor-A humanized mice, the same set of human cytokines (IFN-gamma, IL-10, IL-5, IL-3, GM-CSF, IL-13) was induced in response to the three CARTs, while the degree of induction varied by treatment. In contrast, Donor D-humanized mice showed vastly different responses to each treatment; uCART 2 induced significant levels of IL-6, TNF-beta, MIG/CXCL9, MDC, MCP-3, IL-13, and IL-2. In sum, we show that PBMC-humanized mice capture individual variations in the toxicity and efficacy of uCARTs. This platform can be used to preclinically assess efficacy and persistence of uCARTs as well as used to predict toxicity from the interaction between the host and healthy T cell donor of uCARTs.

SR-18292 Induces Fetal Hemoglobin Synthesis and Reduces Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a commonly inherited blood disorder, which affects millions around the world. Reactivation of fetal hemoglobin (HbF) synthesis in adult erythroid cells can diminish the severity of many clinical features of the disease. Up-regulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to induce HbF levels in human primary erythroid progenitor CD34 + cells. Here we reported the in vitro and in vivo effects of a small molecular compound, SR-18292, on HbF induction via modulating PGC-1α. To determine whether SR-18292 can induce HbF in human CD34 + cells, we performed RT-qPCR, flow cytometry, and western blot to measure the mRNA and protein levels of PGC-1α and γ-globin. We found that mRNA levels of PGC-1α and γ-globin were significantly increased in cells treated with SR-18292 while β-globin mRNA levels were unaffected. Protein levels of γ-globin and the ratio of F-cells were increased in treated cells. Erythroid cellular differentiation markers (CD71/CD235a) and terminal differentiation markers (CD49d/CD233) were used to examine the differentiation status. There was no significant difference in the population of CD71 +CD235a + cells among all groups. However, SR-18292 treatment increased the percentage of CD49d -CD233 + cells at 5 or 7.5 μM. These results suggest that SR-18292 at higher concentrations can promote erythroid terminal differentiation. To evaluate whether the HbF induction by SR-18292 can reduce the SCD disease pathology in SCD mice or not, we intraperitoneally administered SR-18292 to SCD mice. HbF induction was determined by flow cytometric analysis using an anti-HbF antibody. And it was shown that the HbF-high cell population was induced in SCD mice after 4 weeks of treatment. And we used transferrin receptor CD71 and erythroid-specific marker Ter119 to determine whether SR-18292 could affect erythroid differentiation. There was no significant difference in the CD71 +Ter119 + population after 4 weeks of SR-18192 treatment compared with untreated animals. The mRNA levels of γ-globin and PGC-1α were increased in treated mice. And the increase of PGC-1α and γ-globin protein levels were confirmed by western blot. Reticulocyte is a sign of premature RBC destruction (hemolysis), and the SCD mice usually have high reticulocyte counts (>50%). We measured the reticulocyte in the peripheral blood of SR-18292 treated and untreated SCD mice by flow-cytometric analyses and found that reticulocyte was significantly reduced in SR-18292 treated animals (from 55% to 20%) after 4 weeks of treatment. Complete blood cell counts (CBC) showed that SR-18292 treated mice had slightly elevated numbers of red blood cells (RBCs) and more hemoglobin, but lower counts of white blood cells, lymphocytes, and neutrophils. In addition, the RBC distribution width (RDW) was significantly reduced after SR-18292 treatment, indicating that the size of circulating RBCs was more uniform in treated mice. In addition, erythroid cell morphology was evaluated by blood smears with Wright-Giemsa stain. The number of sickled RBCs were reduced in SCD mice treated with SR-18292 for 4 weeks. This is consistent with our observation that RDW was reduced in SCD mice treated with SR-18292. Furthermore, SR-18292 treated mice had a statistically significant reduction in spleen size and weight in contrast to the spleens of untreated SCD mice. The indices of RBCs were improved in accompany with reduced hemolysis and a higher fraction of circulating mature erythrocytes in treated SCD mice. There were no observable adverse effects on other hematopoietic cell lineages. We also performed single-cell RNA-seqs in human CD34 + cells treated with SR-18292 and found that some known HbF repressors (such as BCL11A and ZBTB7A) were decreased in treated groups. Besides that, there were many differentially expressed genes (DEGs), which might be novel HbF regulators. We are verifying those DEGs genes by qPCR and using CRISPR/cas9 to examine their functions in globin regulation. In summary, our findings suggest that SR-18292 might be a promising therapeutic agent to treat SCD and modulating PGC-1α activity or the signaling pathways that it regulates might benefit therapeutically patients with SCD.

Dynamic Oscillation of Tetraspanin CD9 Orchestrates Engraftment and Mobilization of Human Hematopoietic Stem/Progenitor Cells

Background Trafficking of hematopoietic stem/progenitor cells (HSPCs) to and from their bone marrow (BM) niche is pivotal for engraftment and mobilization during clinical transplantation, where both processes are controlled by highly orchestrated molecular programs. We previously identified the tetraspanin CD9 as a downstream effector of the SDF-1/CXCR4 axis driving migration, adhesion and homing of human CD34+ HSPCs, but its consequential impact on stem cell engraftment and mobilization as well as the underlying mechanisms remains unknown. Methods Umbilical cord blood (UCB) CD34+ cells were exposed to a CD9 neutralizing antibody ALB6 prior to infusion into sublethally irradiated NOD/SCID mice, with overall engraftment and lineage output assessed by enumerating human CD45+ (pan-leukocytes), CD19+ (B-lymphoid), CD33+ (myeloid) and CD34+ (stem/progenitors) cells in the recipient BM at 8 weeks post-transplantation. CD9 expression on engrafted HSPCs was characterized by flow cytometry and compared to those before transplantation or after G-CSF mobilization. The oscillation pattern was then validated on HSPCs in pairwise BM, peripheral blood (PB) and G-CSF-mobilized peripheral blood (MPB) samples from healthy donors. Activity of integrin VLA-4 (an established binding partner of CD9) was evaluated on BM-residing and mobilized HSPCs by VCAM-1 binding assay. Results A short exposure of UCB CD34+ cells to the CD9 neutralizing antibody markedly inhibited their BM engraftment by 64.2% ( P=0.003), reflected by the general decline in human CD45+ cell population. There was no bias in lymphoid/myeloid lineage outputs, suggesting CD9-mediated engraftment is not restricted to specific subsets of hematopoietic precursors. CD9 expression was drastically elevated on engrafted CD34+ cells in the BM from 9.6% to 91.4% ( P<0.001) compared to pre-transplantation. In contrast, CD9 expression was significantly downregulated on BM CD34+ cells from 73.4% to 33.5% ( P<0.001) upon G-CSF mobilization, with the decline observed in both primitive stem cells (CD34+CD38-) and committed progenitors (CD34+CD38+), indicating that the oscillation of CD9 is highly associated with HSPC trafficking. Concordantly, pairwise analysis of steady-state HSPCs in BM and PB from healthy donors revealed higher CD9 level in BM-residing CD34+ cells than the PB-circulating portion (46.1% vs. 26.5%, P=0.004; n=13). In addition, a significantly higher CD9 expression was observed on BM CD34+ cells (47.7%; n=16) than those in MPB (32.3%, n=25; P=0.003). CD9 expression on MPB CD34+ cells was enhanced upon SDF-1 exposure ( P=0.006), revealing a dynamic regulation. In normal BM, CD9+ HSPCs exhibited elevated VLA-4 activities with preferential binding to soluble VCAM-1 compared to the CD9- counterpart (51.4% vs. 25.9%, P<0.001; n=10). Superiority in VCAM-1 binding capacity was also witnessed in BM-residing CD34+ cells compared to MPB (37.7% vs. 4.1%, P=0.014), implicating that the influence of CD9 on HSPC trafficking may due to the alteration of integrin activities and downstream adhesive properties. Conclusion This study describes the dynamic regulation of CD9 during entry and exit of HSPCs to and from their BM niche via modulation of integrin activities. The findings extend our understandings on HSPC trafficking that could potentially lead to new strategies to enhance clinical stem cell transplantation and mobilization.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Deciphering the Boundaries of KBTBD4-CoREST1 Axis Modulation to Maximally Expand Human HSCs

Background: The UM171 technology has led to major progress in the expansion of cord blood units (CBUs) with low cellularity (Fares et al., Science 2014). This breakthrough has notably increased availability of better HLA-matched CBUs and improvements in clinical outcome (Cohen et al., Lancet Hematol 2020 and Blood Advances 2023, PMID: 37467030; see also Cohen et al. and Milano et al., this meeting). Enhanced HLA matching is a direct consequence of the selection of CBUs which are otherwise too small without expansion. Indeed, with this technology, approximately 50% of units in CB banks are eligible for clinical use, versus 5% without expansion. As recently reported, UM171 promotes the activation of the novel CUL3-KBTBD4 E3 ligase complex leading to the ubiquitylation and degradation of its natural substrate, CoREST1, a repressor complex that includes RCOR1, LSD1, HDAC1/2. This complex is upregulated in human HSC upon their ex vivo culture where it compromises their potential in the absence of UM171 (Chagraoui et al., Cell Stem Cell, 2021). Aims and Methods: The goal of this research is to understand the boundaries of KBTBD4-CoREST1 axis modulation that determine the optimal expansion of human HSCs in vitro and in vivo. More precisely, we monitored the relationship between CoREST1 levels in primary CD34+CD45RA-CD90+ CB cells and determined the impact of maximal CoREST1 degradation on the yield of expanded HSC using a series of functional in vitro and in vivo approaches along with transcriptomic data. Results: Using flow cytometry, we noticed a gradual degradation of RCOR1 with increasing concentrations of UM171 with a plateau at 125nM suggesting that maximal RCOR1 degradation was reached at this dose which represented a 4-fold increase above the concentration of UM171 used in current clinical manufacturing. Of interest, at high concentration of UM171 (i.e.,125nM), total nucleated cell expansion was reduced by approximately 30% while that of primitive CD34+CD45RA-CD90+EPCR+ HSC was maximal and significantly above that measured at 35nM suggesting a preferential impact of CoREST1 degradation on the more primitive cell compartment along with improvement in graft quality. CITE-Seq experiments confirmed the preferential expansion of primitive HSPC including HSCs and LMPPs with a reduction of more mature cellular subsets when cells were exposed to 125nM of UM171 in comparison to 35nM. The in vivo functionality of the 7-day expanded grafts (35nM vs 125nM UM171) were compared by transplanting the outcome of 100 Day 0 CD34+ CB cells in NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) mice, a cell dose representing less than 1 competitive repopulation unit (CRU) of unexpanded cells in this model. Using this low cell dose (limit dilution), we observed that the 125nM expanded graft provided 10-times greater engraftment than observed in mice transplanted with cells exposed to 35nM UM171 with values reaching a median of 5±5% vs. 0.4±0.13%(n=10 mice per condition, p=0.01; Mann Whitney test) of human engraftment at 12 weeks post-transplantation, respectively. Conclusion: Maximal stimulation of CUL3-KBTBD4 by higher concentration of UM171 leads to much better expansion of functional human HSC and opens new avenues for optimal expansion of very small cord blood units and possibly adult bone marrow-derived HSCs which require more than minimal manipulation (e.g., gene therapy). We believe this new data represents a novel milestone in HSC expansion with obvious subsequent clinical benefits.

Nutrient-Sensitising Drug Repurposing Screen Identifies Lomerizine As a Mitochondrial Metabolism Inhibitor in Chronic Myeloid Leukaemia

In chronic myeloid leukaemia (CML), the persistence of leukaemic stem cells (LSCs) following first line treatment with tyrosine kinase inhibitors (TKIs) such as imatinib, can lead to disease relapse. We have previously demonstrated that therapy-resistant CML LSCs rely on oxidative phosphorylation (OXPHOS) for survival and that targeting mitochondrial metabolism sensitises CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy, or are associated with adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. To identify clinically applicable compounds that target cells dependent on mitochondrial metabolism, we used a nutrient-sensitised repurposing screening strategy. Upon replacement of glucose with galactose as the only sugar source in cell culture media, there is a shift in cellular metabolism towards OXPHOS (as cells are unable to effectively use galactose for glycolysis). Therefore, CML cells grown in galactose-containing media are more sensitive to inhibition of mitochondrial respiration than glucose-grown cells. Using this approach, we screened total of >1,100 unique and structurally diverse FDA-approved drugs in CML cells, cultured in the presence of 11mM glucose or 11mM galactose. This uncovered lomerizine dihydrochloride, a L-type Ca2+ channel blocker used as an anti-migraine drug, as a candidate compound effective against OXPHOS-dependent CML cells. Subsequently, patient derived CML CD34+ cells were shown to have increased ER mass (the main intracellular Ca2+ store) and an increase in mitochondrial Ca2+ levels compared with normal cells. Moreover, transcriptional analysis revealed that Ca2+ channel genes such as voltage-gated CACNA1D and receptor-activated TRPC6 are significantly upregulated in CML LSCs (CD34+CD38-) when compared with normal haematopoietic stem cells (HSCs), which was not reverted following imatinib treatment. Therefore, we hypothesised that the observed increase in mitochondrial metabolism in CML LSCs was dependent on CACNA1D and TRPC6 activity, and the increase in mitochondrial Ca2+ levels, which could be inhibited by lomerizine treatment. Notably, CRISPR-Cas9-mediated CACNA1D or TRPC6 knockout significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and decreased cellular oxygen consumption rate, indicative of reduction in OXPHOS. Similarly, while imatinib treatment did not affect intracellular Ca2+ levels in primary CML CD34+ cells, lomerizine treatment significantly reduced cytoplasmic and mitochondrial Ca2+ levels, and mitochondrial respiration/OXPHOS. Applying liquid chromatography-mass spectrometry and isotope assisted metabolomics, we traced labelled nutrients (glucose and palmitate) in CD34+ cells. This revealed a significant reduction in activity of mitochondria-located Ca2+ dependent dehydrogenases and overall reduction in tricarboxylic acid (TCA) cycle activity in primary CML cells following lomerizine treatment. Of clinical relevance, using combination of cell surface markers, cell division trackers and functional assays, including long-term culture initiating cell (LTC-IC) assay, we demonstrate that lomerizine treatment, alone or in combination with imatinib, targeted CML LSCs when cultured in physiological medium in vitro, without affecting normal HSCs. Notably, combination treatment with imatinib and lomerizine reduced CML tumour burden, targeted CML LSCs and extended survival in xenotransplantation model of human CML. In summary, we reveal for the first time that Ca2+ regulation is disrupted in CML stem cells and influx via CACNA1D and TRPC6 is crucial for maintaining high mitochondrial Ca2+ levels and enhanced mitochondrial OXPHOS. This renders CML stem cells sensitive to lomerizine, an FDA-approved Ca2+ inhibitor identified following metabolism-specific drug-repurposing screening.

Relationships between CD34-, CD105- and bcl-2-Expression Levels and Contrast-Enhanced Ultrasound-Based Differential Diagnosis of Adnexal Tumours

The relationships between CEUS parameters of adnexal tumours and postoperative immunohistochemical assessments of CD34, CD105 and bcl-2 were analysed. This study aimed to investigate whether contrast-enhanced ultrasonography (CEUS) parameters depend on the microvascular density of the tumour lesion found after surgery. Fifty-one patients with a diagnosis of adnexal tumours were included in this single-centre, prospective study. Participants underwent preoperative CEUS (contrast-enhanced ultrasound). Colour Doppler enhancement characterisation parameters (Ystart, Ymax and S) were determined. Immunohistochemical examination of histological specimens of the adnexal lesions was then carried out to determine the expression levels of the CD34, CD105 and bcl-2 proteins. Relationships between the aforementioned parameters were investigated. No significant statistical correlations were observed between CD34, CD105 and bcl2 expression levels and CEUS parameters, independently of whether the operated lesion was malignant or benign. Transvaginal CEUS is diagnostic for the detection of pathological neoplastic vascularisation of an adnexal lesion independent of the density of microcapillaries found postoperatively.

The CD47-Blocking Immune Checkpoint Inhibitor Maplirpacept Does Not Interfere with Blood Transfusion Compatibility Testing

CD47 is a transmembrane glycoprotein that delivers an anti-phagocytic (“do-not-eat”) signal by binding to signal regulatory protein α (SIRPα) on the surface of macrophages. Many tumors express high levels of CD47 as a means to escape macrophage-mediated immune surveillance and blockade of the CD47-SIRPα pathway has emerged as a novel therapeutic strategy with demonstrated anti-tumor activity both in vitro and in vivo (Petrova et al. 2017; Weiskopf 2017). Maplirpacept (PF-07901801, TTI-622) is a soluble human SIRPαFc fusion protein with an IgG4 domain which acts as an innate immune checkpoint inhibitor that prevents CD47-SIRPα binding, engages activating Fcγ receptors on macrophages, and promotes phagocytosis of malignant cells (Lin et al. AACR 2018). Maplirpacept is currently in clinical development to treat patients with ovarian cancer, multiple myeloma, acute myeloid leukemia and lymphoma. Additionally, CD47 is expressed on red blood cells (RBCs) and plays a role in the natural clearance of aged RBCs by splenic macrophages in vivo (Oldenborg et al. 2000). CD47 expression on RBCs was shown to interfere with pre-transfusion laboratory testing in patients treated with the anti-CD47 mAb, Hu5F9-G4 (Velliquette et al. 2019). Unlike anti-CD47 mAbs, minimal binding to human RBCs is observed with Maplirpacept (Lin et al. AACR 2018). In this study we investigated whether the minimal binding of Maplirpacept to human RBCs precludes interference with routine pre-transfusion laboratory tests. RBC suspensions from healthy donors representing all 8 ABO-Rh blood groups were spiked with Maplirpacept at 0.6, 1.2 or 2 mg/ml or anti-CD47 (clone Hu5F9). Blood typing was performed according to the manufacture's protocol (Seraclone, Biorad) and observed for the presence or absence of agglutination. For cross matching experiments, plasma was prepared from blood of healthy donors and spiked with Maplirpacept or anti-CD47 at the same concentrations used for the blood typing experiments. RBCs from two healthy donors were mixed with plasma and an indirect antiglobulin test (IAT) was performed according to the manufacture's protocol (Coombs Anti-IgG, Biorad). Using RBCs from healthy donors representing the 8 major ABO-Rh blood groups, we now show that the addition of Maplirpacept does not cause agglutination of test RBCs and produces the expected blood typing result in a standard forward blood typing test. No interference was observed at the highest concentration tested. However, addition of an anti-CD47 mAb caused pan-agglutination as well as false positive blood typing results (Table 1). In a standard IAT that mimics the in vitro compatibility cross matching test performed prior to a blood transfusion, the anti-CD47 mAb gave false positive results while Maplirpacept produced results identical to the control serum condition (Table 2). This data is consistent with a study demonstrating interference by Hu5F9 in routine pre-transfusion blood testing (Velliquette et al. 2019). Additionally, no interference was observed when Maplirpacept was added to conditions which produced agglutination in a standard IAT across a range of blood types. Interference with RBC panel testing has important consequences in transfusion medicine as the presence of irregular antibodies can complicate the selection of suitable RBC units for treated patients. Here we demonstrate that interference with blood compatibility testing is dependent upon the type of CD47-blocking agent. Decoy receptors such as Maplirpacept, unlike anti-CD47 mAb, do not interfere with blood group serologic testing, which may confer a significant clinical advantage by negating the need for additional approaches to prevent interference.

Characterization of Multiple Myeloma Subpopulations Associated with Treatment Resistance By Single-Cell RNA Sequencing

Multiple myeloma (MM) has extensive genomic and transcriptomic heterogeneity with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire). Even though new treatment approaches have been developed, most cases of MM remain incurable. Recent evidence suggests that epigenetic cell plasticity and changes in cell state underlie the therapy resistance, but the mechanisms remain largely unknown. We attempted to understand the comprehensive architecture of the entire tumor cell populations using repertoire clonality as a fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were present at very low frequencies in some minor fractions other than the main fraction (Lineage marker - (Lin -)/CD19 -/CD38 +/CD138 +) as the disease stage progressed. Importantly, MM cells were consistently present in CD138 negative fraction (Lin -/CD19 -/CD38 +/CD138 -) regardless of disease stage. Flow cytometry (FCM) analysis confirmed that CD138negative MM cells were present at a rate of less than 10% across the disease stages. Compared with CD138 positive cells, they were slowly cycling and showed unique morphology with large cytoplasm (n=59). To achieve a selective and comprehensive evaluation of MM population, we performed single-cell RNA sequencing (scRNA-seq) coupled with VDJ targeted sequencing (scVDJ-seq) on Lin -/CD19 -/CD38 +(without CD138 gating) samples from 11 MM patients, including those at diagnosis and in remission with minimal residual disease (MRD). This method allowed us to discriminate MM cells (20,084 cells) clearly from others by clonal repertoire sequence even in the post-treatment samples with extremely small populations of tumor cells. Because mRNA levels of CD138 did not correspond to the differential intensity in FCM, we defined gene signatures of CD138 positive and negative cells respectively by bulk RNA-seq (n=6). As a result of integration of these gene signatures using Gene Set Enrichment Analysis (GSEA), the heterogeneity on the CD138 axis was also unraveled in scRNA-seq data. Interestingly, gene ontology (GO) analysis of bulk RNA-seq data revealed that several terms related to H3K4 methylation were positively enriched in CD138 negative fraction, which included KMT2A, KMT2B, KMT2C and KMT2D. Western blotting confirmed significant reductions in H3K4me1 and H3K4me3 levels in CD138 negative cells. Furthermore, primary MM cell culture indicated the plasticity between CD138 positive and negative cells, which was also suggested by trajectory/velocity analysis of scRNA-seq data. Collectively, it was suggested that epigenetic changes, including those in histone modifications, contribute to the heterogeneity along the CD138 axis. We next compared the size of each cluster before and after treatment in scRNA-seq data to identify the MM populations associated with treatment resistance. Remarkably, we identified unique clusters which tended to remain at high proportions against treatment in the CD138 negative population. They were characterized by low expression of MHC class-I genes and TNFRSF17/BCMA, as well as 198 significantly upregulated genes including MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance of MM. To systematically interrogate their contribution in MM cell survival, we performed CRISPR/Cas9 knockout screening of selected 100 genes, which were differentially up-regulated in the resistant clusters and associated with poor prognosis in a MMRF CoMMpass Study database. GO analysis of indispensable genes reproducibly identified among three MM cell lines revealed enrichment of several pathways related to mRNA splicing. These results suggested that the RNA splicing pathway is a promising therapeutic target to eliminate resistant populations. Indeed, we confirmed that inhibition of a splicing factor by CRISPR/Cas9-mediated knockout and pharmacological protein degradation suppresses MM cell proliferation effectively. Taken together, we achieved a complete view of MM diversity by using repertoire clonality and confirmed heterogeneity along the CD138 axis, which may be characterized by epigenetic plasticity. Our results highlight a novel subpopulation as a candidate therapeutic target for cure.

T Cell Dysfunction and Exhaustion in Patients with CLL: The Impact of Long Term Ibrutinib Treatment

Introduction: Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with defects in the immune system and is dependent on the microenvironmental niche for survival. Ibrutinib, the first irreversible inhibitor of Bruton's tyrosine kinase targets B-cell receptor signaling to kill tumor cells. In addition, ibrutinib also appears to regulate the tumor microenvironment and T-cell immunity, but this mechanism unknown. Increased expression of inhibitory receptors, and functional defects such as reduced proliferative capacity and cytotoxicity are common in T cells from patients with CLL. The effect of ibrutinib on T-cell immunity has not been extensively studied in the setting of long term ibrutinib treatment. Therefore, we investigated the phenotype and function of CD4+ and CD8+ T-cells from patients with CLL treated with ibrutinib, treatment naïve CLL and age-matched healthy controls. Methods: CLL patient samples were obtained from treatment naïve patients at diagnosis, and at the 3- and 5-year timepoints. Samples from patients on continuous ibrutinib who had not relapsed at the 3- and 5-year timepoints; and pre-ibrutinib were obtained as well. Age matched healthy donor-derived peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll. PBMCs from CLL samples and healthy donors were labeled with CellTrace Violet and treated with anti-CD3 and anti-CD28 (ThermoFisher) for 7 days. T-cells were stimulated to identify proliferation capacity and to identify immune profiles. Results: Efficient T-cell activation and proliferation requires multiple signals such as T-cell receptor (TCR) stimulation, a costimulatory signal, and cytokine production. We analyzed the proliferation of T cells in-vitro upon stimulation with anti-CD3/CD28 beads using CellTrace Violet on PBMCs and found that this stimulation resulted in increased expansion of CD4+ T cells and CD8+ T cells in all the samples. We also found significant difference in the proliferation between untreated and treated CLL samples. Interestingly, proliferating T cells had different expression pattern of the exhaustion markers and inhibitory molecules such as TIM3, TIGIT, LAG3, CD160, KLRG1, CD244 and PD-1 at different phases of the disease. Increased expression of CD160, CD244, and PD1 are the key features of T-cell exhaustion. We found significant increase in the percentage of T cells from CLL patients expressing CD244, CD160 and PD1 compared to healthy donor controls but not between naïve and ibrutinib treated samples. Long term ibrutinib treatment in 3yr and 5yr samples does not change the exhaustion state in patients. We also found T-cells from ibrutinib treated samples express higher levels of CD57 which is known as a marker of senescence (Figure 1A). The phenotypes of untreated CLL samples are CCR7+CD45RA- central memory T-cells (35%) and CCR7+CD45RA+ naïve T cells (25%) (p<0.05) after stimulation in-vitro on day 3. In the same setting, ibrutinib treated samples had higher levels of effector memory T-cells (p<0.05) (80%) compared to untreated CLL samples (Figure 1B). These results suggest that the phenotype has changed to central memory phenotype in untreated samples. Functional T-cell exhaustion in CLL also leads to failure in cytokine production. Therefore, we measured different cytokine levels after stimulation in all the patient samples and in healthy controls. We found significant increase in the levels of inflammatory cytokines such as IFN-γ (p<0.05) and TNF-α (p<0.05) in long term ibrutinib treated samples compared to naïve CLL samples and healthy controls. Conclusion: Several studies have showed that T-cell counts are increased after treatment with ibrutinib in patients with CLL but not in extended therapy. Our findings suggest that long term ibrutinib treatment does increase T-cell number and proliferative capacity in CLL. The proliferating T cells express higher levels of exhaustion markers and inhibitory molecules in-vitro making them dysfunctional. However, these T-cells still maintained good proliferative potential, on par to healthy donors and better than untreated patients which can be applied in expanding improved T cell grafts for various types of adoptive T cell therapy, such as TCR gene therapy, and CAR therapy. Also better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells.

Breaking Barriers in B-Acute Lymphoblastic Leukemia Treatment: Experience of a Tertiary Hospital with the Use of a Tandem Dual CAR-T 19/22 in Pediatric & AYA Patients

INTRODUCTION Anti-CD19 chimeric antigen receptor (CAR) autologous T-cell therapy tisagenlecleucel (CTL019) has shown efficacy in pivotal trials and real-world data in relapsed/refractory pediatric B-cell precursor acute lymphoblastic leukemia (r/r B-ALL). However, 30-50% of patients relapse, because of CD19 loss in leukemic cells, or persistence/function reduction of CTL019. To improve relapse free survival, multitargeting CAR-T cells, such as bicistronic anti-CD22 and anti-CD19 or single sequential infusion are being developed. We report the safety and efficacy of an in-house manufactured tandem anti-CD19/22 dual CAR-T cells in pediatric, adolescents and young adults (AYA) diagnosed with r/r B-ALL. MATERIAL AND METHODS Eight batches of the tandem anti-CD19/22 dual CAR-T cells were manufactured under Good Manufacturing Practices (GMP) and authorized under a compassionate use program by the Spanish Agency for Medicines and Health (AEMPS). The starting material in all cases was patient´s non mobilized apheresis. After, CD4 + and CD8 + T cells were immune selected, activated with CD3/CD28 beads and transduced with a CD19/CD22 lentiviral vector (from Miltenyi Biotec®). Cell expansion was performed in a CliniMACS Prodigy® device with TexMACs® culture medium supplemented with IL-7 and IL-15. During manufacturing, process and quality controls were carried out and the finished product was infused fresh or cryopreserved. RESULTS A total of 8 patients with a median age of 10.5 (range 6-25) years old received a median of 1 (range 1-3) doses of tandem anti-CD19/22 dual CAR-T cells containing a median of 1.13x106 (range 7.5x105-3x106) cells/kg infused. First 3 patients (37.5%) received cryopreserved product. Two patients (25%) were also diagnosed with Down syndrome (DS). The median time between apheresis and infusion was 10.5 days (range 9-14 ). Five patients (62.5%) received previously tisagenlecleucel and four of them (50%) had also received a hematopoietic stem cell transplantation (HSCT). Leukemia blasts did not express CD19 in 2 patients (25%). Both received tisagenlecleucel previously, and one of them also expressed low levels of CD22. Median pre-infusion minimal residual disease (MRD) was 4.91% (range 0.01-87). Bridging therapy was low dose chemotherapy in 7 patients (87.5%), using inotuzumab in 1 patient. Lymphodepletion regimen was individualized, in 5 patients (62.5%) consisted on fludarabine 30 mg/m2/day for 4 days and cyclophosphamide 600 mg/m2/day for 2 days. Regarding CAR cell product, mean transduction was 41.4 %. Similar CD4 +/CD8 + distribution was seen in most products generated (7/8, 88%), and central memory cells were the main subpopulation found, followed by naïve cells. Most CAR-T cell expansions (88%, median 7, range 7-28) were seen between day +7 and day +21 post-infusion. Regarding toxicity, 7 patients (87.5%) presented cytokine release syndrome (CRS). Three patients (37.5 %) with high pre CAR-T tumor burden (TB) (range 75-87 %) developed CRS grade 3-4 (ASBMT guidelines) followed by ICANS grade 3. On day +28 after infusion, 6 patients (75%) were in complete remission (CR), one of them with positive minimal residual disease (MRD). Two patients died because of disease progression (range 2-4.5 months) and a third one (with DS) because of toxicity before day +28 after CAR-T infusion. Consolidative HSCT was performed in 3 of 6 patients that achieved CR (50%) during the first 90 days after CAR-T cell infusion. During follow up of these transplanted patients (median 8.4 months, range 2.3-12.3), 1 patient (33.3%) relapsed after 11 months. One patient presented central nervous system relapse 1 month after CAR-T infusion and HSCT was performed and with 35 months of follow up, continues alive in CR. The last patient (with DS) relapsed 3 months after infusion and immunotherapy (inotuzumab) has been given in order to perform a second HSCT if CR is achieved. CONCLUSIONS In-house manufacturing of tandem anti-CD19/22 dual CAR-T cells is feasible and reproducible under GMP conditions in an academic institution. Product infusion seems to be well tolerated in most patients, although patients with high TB developed high grade toxicities. Setting bridging therapy, reducing TB and manufacturing time remains a challenge. Our experience suggests the efficacy of its use as a bridge to consolidative HSCT. Nevertheless, a larger number of patients in the context of a clinical trial would be necessary.