Highlights d Patients with severe COVID-19 accumulate HLA-DR Low monocytes and immature neutrophils in blood/lungs d Calprotectin level positively correlates with neutrophil count and disease severity d Loss of non-classical monocytes could identify high risk of severe COVID-19

Summary Accumulation of CD103 + CD8 + resident memory T (T RM ) cells in human lung tumors has been associated with a favorable prognosis. However, the contribution of T RM to anti-tumor immunity and to the response to immune checkpoint blockade has not been clearly established. Using quantitative multiplex immunofluorescence on cohorts of non-small cell lung cancer patients treated with anti-PD-(L)1, we show that an increased density of CD103 + CD8 + lymphocytes in immunotherapy-naive tumors is associated with greatly improved outcomes. The density of CD103 + CD8 + cells increases during immunotherapy in most responder, but not in non-responder, patients. CD103 + CD8 + cells co-express CD49a and CD69 and display a molecular profile characterized by the expression of PD-1 and CD39. CD103 + CD8 + tumor T RM , but not CD103 − CD8 + tumor-infiltrating counterparts, express Aiolos, phosphorylated STAT-3, and IL-17; demonstrate enhanced proliferation and cytotoxicity toward autologous cancer cells; and frequently display oligoclonal expansion of TCR-β clonotypes. These results explain why CD103 + CD8 + T RM are associated with better outcomes in anti-PD-(L)1-treated patients.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ~40% in chronic myelomonocytic leukemia (CMML). ASXL1 mutations associate with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients with shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+; Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global level of H3K27ac, and Flt3 upregulation. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands, PD-L1/L2, CD155, and CD80/86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or shRNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wildtype T cells overexpressed PD-1 and TIGIT receptors, leading to a predominant exhausted T cell phenotype. Combined inhibition of MEK and BET led to downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8+ T cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6–CTTN axis as a positive regulator of human but not mouse MK maturation.

Gut dysbiosis has been associated with intestinal and extraintestinal malignancies, but whether and how carcinogenesis drives compositional shifts of the microbiome to its own benefit remains an open conundrum. Here, we show that malignant processes can cause ileal mucosa atrophy, with villous microvascular constriction associated with dominance of sympathetic over cholinergic signaling. The rapid onset of tumorigenesis induced a burst of REG3γ release by ileal cells, and transient epithelial barrier permeability that culminated in overt and long-lasting dysbiosis dominated by Gram-positive Clostridium species. Pharmacologic blockade of β-adrenergic receptors or genetic deficiency in Adrb2 gene, vancomycin, or cohousing of tumor bearers with tumor-free littermates prevented cancer-induced ileopathy, eventually slowing tumor growth kinetics. Patients with cancer harbor distinct hallmarks of this stress ileopathy dominated by Clostridium species. Hence, stress ileopathy is a corollary disease of extraintestinal malignancies requiring specific therapies. Significance: Whether gut dysbiosis promotes tumorigenesis and how it controls tumor progression remain open questions. We show that 50% of transplantable extraintestinal malignancies triggered a β-adrenergic receptor–dependent ileal mucosa atrophy, associated with increased gut permeability, sustained Clostridium spp.–related dysbiosis, and cancer growth. Vancomycin or propranolol prevented cancer-associated stress ileopathy. This article is highlighted in the In This Issue feature, p. 873

The p210bcr-abl protein was shown to inhibit apoptosis induced by DNA damaging agents. Apoptotic DNA fragmentation is delayed in the bcr-abl+ K562 and KCL-22 compared with thebcr-abl− U937 and HL-60 cell lines when treated with etoposide concentrations that induce similar DNA damage in the four cell lines. By the use of a cell-free system, we show that nuclei from untreated cells that express p210bcr-abl remain sensitive to apoptotic DNA fragmentation induced by triton-soluble extracts from p210bcr-abl− cells treated with etoposide. In the four tested cell lines, apoptotic DNA fragmentation is associated with a decreased expression of procaspase-3 (CPP32/Yama/apopain) and its cleavage into a p17 active fragment, whereas the long isoform of procaspase-2 (ICH-1L) remains unchanged and the poly(adenosine diphosphate–ribose)polymerase protein is cleaved. These events are delayed in bcr-abl+ compared with bcr-abl− cell lines. The role of p210bcr-abl in this delay is confirmed by comparing the effect of etoposide on the granulocyte-macrophage colony-stimulating factor (GM-CSF)–dependent UT7 cells and thebcr-abl–transfected GM-CSF–independent UT7/9 clone. We conclude that the cytosolic pathway that leads to apoptotic DNA fragmentation in etoposide-treated leukemic cells is delayed upstream of procaspase-3–mediated events in bcr-abl+ cell lines.

Context. The perhaps only CMML-specific Randomized Clinical Trial (RCT) established hydroxyurea (HY) as the main treatment (Tx) for advanced proliferative CMML (Wattel Blood 1996). In Europe, the only hypomethylating agent (HMA) approved in CMML is AZA in non proliferative CMML-2. Phase 2 trials reported the activity of decitabine (DAC) in advanced proliferative CMML (Braun Blood 2011, Santini Leukemia 2018). We performed a RCT of DAC (±HY during the first 3 cycles) vs HY alone in those pts. Methods. The DACOTA trial (EudraCT 2014-000200-10) accrued pts with previously untreated (or < 6 weeks of HY), proliferative (WBC ≥ 13x109/L) CMML with advanced disease defined per Wattel et al as presence of extramedullary disease or ≥2 criteria among: BM blasts ≥5%, abnormal karyotype (except -Y), ANC ≥ 16x109/L, Hb < 10 g/dL, platelets < 100 x109/L or splenomegaly > 5 cm below costal margin. Pts were randomized 1:1 to DAC (20 mg/m2/d IV 5d/28d) or HY (1g/d, adjusted on WBC, 28d cycles) and treated until death, AML transformation or progression. The primary endpoint was EFS, events being death, transformation to AML, progression of myeloproliferation after 3+ cycles or progression of blasts and cytopenias after 6+ cycles. Response was assessed with IWG 2006 criteria modified to account for improvement of myeloproliferation, after central morphology review. Intent-to-treat analyses were done considering missing responses as failures. Results. From Oct 2014 to Sep 2019, 217 pts from 47 centers were screened and 170 randomized (84 DAC and 86 HY), including 12 pts (6 DAC and 6 HY) who never started Tx. Median age was 73 years (IQR 68-78). WHO was CMML-NA/1/2 in 2, 114 and 54 pts, respectively (resp). Median WBC 34.9 x109/L (IQR 22.9-55.7). Cytogenetic risk (Such Haematologica 2011) was fav 69%, int 12%, adv 18% NA 1%. Mutations in TET2, SRSF2, ASXL1 and signaling genes (CBL, JAK2, FLT3, KIT, NRAS, KRAS and CSF3R) were present in 64%, 51%, 62% and 57% resp. 72 pts had received HY for a median 27 days prior to randomization. Aside from older age in the HY arm (median 74 vs 71.5y in the DAC arm), there was no imbalance between Tx arms. DAC and HY pts received a median of 5 (IQR 3-12) and 6 (IQR 3-14) cycles, resp. As of 15th June 2020, 5 and 10 DAC and HY pts were still on Tx. Reasons for Tx cessation in the DAC arm were death (n=19), AML transformation (n=16), progression (n=9) , hematological toxicity (n=13) or other (n=21). Reasons for Tx cessation in the HY arm were death (n=14), AML transformation (n=13), progression (n=18), hematological toxicity (n=6) or other (n=20). 126 and 85 pts received 3 and 6 cycles, resp. In the ITT population, ORR at 3 cycles was 56% (7CR, 25 mCR±HI, 15 SD+HI) and 30% (0 CR, 8 mCR±HI, 18 SD+HI) in the DAC and HY arms, resp (p=0.0011) and ORR at 6 cycles was 32% (6 CR, 9 mCR±HI, 12 SD+HI) and 17% (2 CR, 4 mCR±HI, 9 SD+HI) in the DAC and HY arms, resp (p=0.033). Median response duration was 15.9 vs 18.2 months (mos) in the DAC and HY arm, resp (p=0.81). Infection and hemorrhage occurred at least once in 49% and 31% of pts, resp. 55% of DAC pts and 38% of HY pts required hospitalization at least once (p=0.05). Non-heme ≥ grade 2 AEs occurred in 79% and 63% of DAC and HY arms, resp (p=0.03). Grade ≥3 cardiac AEs occurred in 13 DAC and 4 HY pts, resp. With a median follow-up of 13.9 mos, median EFS was 12.6 vs 10.3 mos in the DAC and HY arms, resp (reference DAC arm, HR= 1.14 CI95 0.8-1.64, p= 0.46). Median AML-free survival (AMLFS) was 13.6 and 15.8 mos in the DAC and HY arms resp (p=0.86). Median OS was 18.4 and 23.1 mos in the DAC and HY arms, resp (p=0.72). Considering death and AML transformation as competing risks there was no significant difference in cumulative incidence of AML (p=0.1) or death without transformation (p=0.06) between arms. 30 pts from the HY arm received an HMA (DAC n= 13, AZA n= 16, both=1) after study exit. Censoring at HMA onset in the HY arm, median OS was 18.4 vs 30.4 in the DAC and HY arm, resp (p=0.15). 13 pts were transplanted (DAC n= 10, HY n= 3). There was no interaction between Tx arm and CMML-0/1 vs -2, platelets ≥ vs <100 x109/L and anemia (Hb < 8 g/dL or RBC-TD vs Hb ≥8) on both EFS and OS (all p>0.05). Conclusion. RCTs are feasible in advanced proliferative CMML, which remains an unmet medical need. In these pts, DAC did not provide an overall or event-free survival advantage over HY. HY remains a valid option in advanced proliferative CMML. However, one third of HY pts subsequently received an HMA and more DAC pts achieved a response and were bridged to HSCT. Figure Disclosures Itzykson: Abbvie: Honoraria; Daiichi Sankyo: Honoraria; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Sanofi: Honoraria; BMS (Celgene): Honoraria; Janssen: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stemline: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncoethix (now Merck): Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees. Santini:BMS, J&J, Novartis: Honoraria; Acceleron, BMS, Menarini, Novartis: Consultancy; Takeda, Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding. Lionel:Abbvie: Consultancy; Takeda: Consultancy; Celgene/BMS: Consultancy, Research Funding; Novartis: Consultancy; Jazz: Consultancy, Research Funding. Thepot:astellas: Honoraria; novartis: Honoraria; sanofi: Honoraria; celgene: Honoraria. Giagounidis:AMGEN: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Luebbert:Janssen: Research Funding. Park:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Other: Travel expenses. Stamatoulas Bastard:Pfizer: Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Celgene: Honoraria; Takeda: Consultancy. Solary:Janssen: Research Funding. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Amgen: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Geron: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Fenaux:Novartis: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Jazz: Honoraria, Research Funding. OffLabel Disclosure: Decitabine for CMML with WBC > 13 x109/L.