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Multiple Myeloma is driven by clonal plasma cell (cPC)-intrinsic factors and changes in the tumorigenic microenvironment (TME). To investigate if residual polyclonal PCs (pPCs) are disrupted, single-cell (sc) RNAseq and sc B-cell receptor analysis were applied in a cohort of 46 samples with PC dyscrasias and 21 healthy donors (HDs). Out of n=234,789 PCs, 64,432 were genotypically identified as pPCs with frequencies decreasing over different disease stages, from 23.66% in monoclonal gammopathy of undetermined significance (MGUS) to 3.23% in MMs (p=0.00012). Both cPCs and pPCs had a comparable expression of typical lineage markers (i.e. CD38, CD138), while others were more variable (CD27, ITGB7). Only cPCs overexpressed oncogenes (e.g. CCND1/2, NSD2), but CCND3 was often expressed in pPCs. BCMA was expressed on both p- and cPCs, while GPRC5D was mostly upregulated in cPCs with implications for on-target, off-tumor activity of targeted immunotherapies. In comparison with HDs, pPCs from patients showed upregulated autophagy and disrupted interaction with TME. Importantly, interferon related pathways where significantly enriched in pPCs from patients vs HDs (p-adjusted < 0.05) showing an inflamed phenotype affecting genotypically normal PCs. Function of pPCs was consequently impacted and correlated with immunoparesis, driven by disrupted cellular interactions with TME. Leveraging our scRNAseq data, we derived a "healthy PC signature" that could be applied to bulk transcriptomics from the CoMMpass dataset and predicted significantly better PFS and OS (log rank p < 0.05 for both). Our findings show that genotypic, single-cell identification of pPCs in PC dyscrasias has relevant pathogenic and clinical implications.

The transglutaminase coagulation factor XIII (FXIII) is critical for the stability and function of intravascular fibrin clots. Pro-repair extravascular fibrin(ogen) deposits are potentially subject to crosslinking by FXIII as well as other transglutaminases not typically resident in plasma. However, the impact of these alternative modifiers on fibrin(ogen) structure and function is not known. We tested the hypothesis that tissue transglutaminase (TG2) modifies FXIII-directed fibrin(ogen) crosslinking in vitro and within injured tissue. Global proteomic analysis following experimental acetaminophen (APAP)-induced acute liver injury revealed that intrahepatic fibrin(ogen) deposition was associated with hepatic TG2 levels that exceeded that of FXIII. Mass spectrometry-based crosslink mapping of in vitro fibrin matrices uncovered the first evidence of synergistic fibrin(ogen) a-a crosslinking catalyzed by both transglutaminases. Fibrin(ogen) crosslinking was increased in livers from patients with APAP-induced acute liver failure. APAP-challenged TG2-/- mice displayed an altered pattern of FXIII-dependent fibrin(ogen)-g and fibrin(ogen)-a chain crosslinking aligned with the impact of TG2 on fibrin crosslinking in vitro. This shift in fibrin(ogen) crosslinking exacerbated pathologies including hepatic necrosis and sinusoidal congestion. The results are the first to indicate that TG2 impacts FXIII-directed fibrin(ogen) crosslinking, both in vitro and in vivo. The results suggest that TG2 functions to dynamically alter the structure of extravascular fibrin(ogen) to mitigate liver damage, a novel mechanism likely applicable across types of tissue injury.

Despite the approval of several new treatments for patients with B-cell lymphoma, there is still a large unmet need. Diffuse large B-cell lymphoma (DLBCL) and Follicular lymphoma (FLs) are the two most common B-cell lymphoma subtypes, accounting for approximately 50% of all cases. EZH2 heterozygous gain-of-function somatic driver mutations are frequently found in Germinal Center B cell (GCB) DLBCLs and FLs. An EZH2 inhibitor has shown durable responses in patients with relapsed or refractory FL, however a considerable fraction of the patients did not show an objective response. To identify alternative therapeutic strategies, we performed CRISPR/Cas9 knockout screens in B-cell lymphoma cells treated with or without an EZH2 inhibitor. This led to the identification of the histone methyltransferase DOT1L as a potential therapeutic target. Specifically, we showed that an EZH2 inhibitor synergizes with a DOT1L inhibitor in a panel of B-cell lymphoma cell lines, regardless of EZH2 mutation status. Mechanistically, we demonstrated that the two inhibitors cooperatively suppress DOT1L-regulated cell cycle genes, up-regulate genes involved in interferon signaling including antigen presenting genes and ultimately drive B cell differentiation by de-repressing EZH2-regulated plasma cell signature genes. Furthermore, we demonstrated the effectiveness of this epigenetic combination strategy in a xenograft model, which resulted in significant abrogation of tumor growth. Together, our studies provide pre-clinical proof-of-concept for an epigenetic combination therapy to overcome resistance and improve durability of response for the treatment of B-cell lymphoma, warranting clinical investigation and illustrating an important convergent role of EZH2 and DOT1L in B cell lymphomagenesis.

Discontinuation of lenalidomide maintenance post autologous transplantation (ASCT) is a burning question within the myeloma (MM) community, especially after the inclusion of MRD in the disease response criteria. In this prospective study, we evaluated the conversion to MRD positivity, the treatment-free survival (TFS) and the progression-free survival (PFS) in 52 MM patients, who discontinued lenalidomide maintenance, after achievement of sustained bone marrow and imaging MRD negativity for three years. Patients who developed MRD positivity after lenalidomide discontinuation, restarted lenalidomide maintenance at the same dose. The median follow-up from lenalidomide discontinuation was 3 years. Overall, 12 (23%) patients obtained MRD positivity and restarted lenalidomide maintenance. Only four (7.6%) patients progressed; 3 had a biochemical progression and one had a clinical progression. The overall median PFS was not reached, while the 7-year PFS from diagnosis was 90.2%. The 1-, 2- and 3-year TFS rates were 93.9%, 91.6% and 75.8%, respectively, whereas the 1-, 2- and 3-year landmark PFS rates from maintenance discontinuation (study entrance) were 96.0%, 96.0% and 92.9%, respectively. There were no statistically significant associations between age, gender, R2-ISS, type of induction therapy and use of consolidation therapy and the effect outcomes of PFS, TFS. We conclude that MRD discontinuation after 3-year sustained marrow and imaging MRD negativity is associated with low rates of MRD conversion and progressive disease. Thus, in the era of modern anti-myeloma treatments, a subgroup of patients may remain treatment free while in complete remission, without jeopardizing disease response.

T-cell recruiting bispecific antibodies (BsAbs) are in clinical development for relapsed/refractory acute myeloid leukemia (AML). Despite promising results, early clinical trials have failed to demonstrate durable responses. We investigated whether activation of the innate immune system through stimulator of interferon genes (STING) can enhance target-cell killing by a BsAb targeting CD33 (CD33 BiTE® molecule, AMG 330). Indeed, we show that cytotoxicity against AML mediated by AMG 330 can be greatly enhanced when combined with the STING agonist 2',3'-cyclic GMP-AMP (cGAMP), or diABZI. We used in vitro cytotoxicity assays, immunoblotting, transcriptomic analyses, and extensive CRISPR-Cas9 knockout experiments to investigate the enhancing effect of a STING agonist on the cytotoxicity of AMG 330 against AML. Importantly, we validated our findings with primary AML cells, and in a xenograft AML model. Mechanistically, in addition to direct cytotoxic effects of STING activation on AML cells, activated T cells render AML cells more susceptible to STING activation through their effector cytokines interferon-gamma (IFNγ) and tumor necrosis factor (TNF), resulting in enhanced type I interferon production and induction of interferon-stimulated genes. This feeds back to the T cells, leading to a further increase in effector cytokines and an overall cytotoxic T-cell phenotype, contributing to the beneficial effect of cGAMP/diABZI in enhancing AMG 330-mediated lysis. We established a key role for IFNγ in AMG 330-mediated cytotoxicity against AML cells, and in rendering AML cells responsive to STING agonism. Here, we propose to improve the efficacy of CD33-targeting BsAbs by combining them with a STING agonist.

Microbial dysbiosis and metabolite changes in the gastro-intestinal (GI) tract have been linked to pathogenesis and severity of many diseases, including graft-versus-host disease (GVHD), the major complication of allogeneic hematopoietic stem cell transplantation (HCT). However, published studies have only considered the microbiome and metabolome of excreted stool and do not provide insight into the variability of microbial community and metabolite composition throughout the GI tract or the unique temporal dynamics associated with different gut locations. Because such geographical variations are known to influence disease processes, we utilized a multi-omics approach to characterize the microbiome and metabolite profiles of gut contents from different intestinal regions in well-characterized mouse models of GVHD. Our analysis validated analyses from excreted stool, but importantly, uncovered new biological insights from the microbial and metabolite changes between syngeneic and allogeneic hosts that varied by GI location and time after transplantation. Our integrated analysis confirmed the involvement of known metabolic pathways, including SCFA synthesis and bile acid metabolism, and identified additional functional genes, pathways, and metabolites, such as amino acids, fatty acids, and sphingolipids, linked to GI GVHD. Finally, we validated a biological relevance for one such newly identified microbial metabolite, phenyl lactate, that heretofore had not been linked to GI GVHD. Thus, our analysis of the geographic variability in the intestinal microbiome and metabolome offers new insights into GI GVHD pathogenesis and potential for novel therapeutics.

The GIMEMA LAL2317 protocol investigated the frontline chemotherapy-blinatumomab combination in adult Philadelphia- CD19+ B-lineage acute lymphoblastic leukemia (Ph- B-ALL) to improve minimal residual disease (MRD) response and clinical outcome. Two cycles of intravenous blinatumomab were administered after chemotherapy cycles 3 and 6. The primary endpoint was the rate of molecular MRD negativity following blinatumomab 1. One hundred and forty-nine patients were enrolled (median age 41 years, range18-65); 132 entered remission, 122 received blinatumomab and 109 had a pre- and post-blinatumomab 1 MRD assessment. MRD negativity increased from 72% to 93% (P<0.001) after blinatumomab, with 23/30 MRD+ patients (73%) becoming MRD-, fulfilling the primary endpoint. At a median follow-up of 38.1 months (0.5-62.8), the median overall and disease-free survivals (OS, DFS) were not reached and the estimated 3-year OS and DFS were 71% and 65%, with an excellent outlook for the patients aged 18-40 years who achieved an early MRD negativity (DFS 92%). Pre-blinatumomab MRD predicted a worse outcome, especially in genetics high-risk patients. Notably, the 3-year survival of blinatumomab-treated patients was 82%. Survival and relapse rates were 91% and 15% in patients assigned to standard chemotherapy, 59% and 35% in patients assigned to HSCT, and 69% and 19% in transplant recipients. Blinatumomab toxicity was manageable, with only 8 permanent discontinuations. This chemotherapy-blinatumomab risk-oriented program yielded remarkable results that need further improvement in higher-risk patients displaying early MRD persistence. Blinatumomab should be considered as a standard component of induction/consolidation for adult Ph- B-ALL. ClinicalTrials.gov # NCT03367299.