Summary The molecular complexity of the bone marrow (BM) microenvironment and its response to stress are incompletely understood, despite its key role in the regulation of hematopoiesis. Here we map the transcriptional landscape of BM vascular, perivascular, and osteoblast niche populations at single-cell resolution at both homeostasis and under stress hematopoiesis. This analysis revealed a previously unappreciated level of cellular heterogeneity within the BM niche, identified novel cellular subsets, and resolved cellular sources of pro-hematopoietic growth factors, chemokines, and membrane-bound ligands. Under conditions of stress, our studies revealed a significant transcriptional remodeling of these niche elements, including an adipocytic skewing of the perivascular cells. Among the stress-induced changes, we observed that vascular Notch ligand delta-like ligands (Dll1,4) were downregulated. In the absence of vascular Dll4, hematopoietic stem cells (HSC) prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the BM niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress, and illustrate the utility of single cell transcriptomic data in systematically evaluating the regulation of hematopoiesis by discrete niche populations.

Purpose: Long peptides are efficiently presented to both CD4 þ and CD8 þ T cells after intracellular processing by antigen-presenting cells. To investigate the safety and in vivo immunogenicity of synthetic overlapping long peptides (OLP) from a human tumor self-antigen, we conducted a phase I clinical trial with OLP from cancer-testis antigen NY-ESO-1 in various adjuvant combinations. Experimental Design: Twenty-eight patients with advanced ovarian cancer in second or third remission were enrolled sequentially in three cohorts and received at least one vaccination. Patients in Cohort 1 (n ¼ 4) received 1.0 mg OLP, Cohort 2 (n ¼ 13) received OLP in Montanide-ISA-51, and Cohort 3 (n ¼ 11) received OLP þ 1.4 mg Poly-ICLC in Montanide-ISA-51 on weeks 1, 4, 7, 10, and 13. Humoral and cellular responses were evaluated by standardized immunomonitoring techniques (ELISA, ELISPOT assay, intracellular cytokine staining, and tetramer staining).Results: The vaccine was generally well tolerated with injection site reactions and fatigue that resolved. NY-ESO-1-specific antibody and CD8þ T cells were undetectable after vaccination with OLP alone, but were found in 6 of 13 (46%) and 8 of 13 (62%) patients, respectively, after vaccination with OLPþMontanide, and in 10 of 11 (91%) and 10 of 11 (91%) patients, respectively, after vaccination with OLPþMontanideþPoly-ICLC. NY-ESO-1-specific CD4 þ T cells were detected in all patients with greater frequency and polyclonality when Montanide-ISA-51 was used for vaccination. Inclusion of Poly-ICLC as an adjuvant further accelerated the induction of NY-ESO-1-specific immune responses.Conclusions: The current study shows that NY-ESO-1 OLP vaccine is safe and rapidly induces consistent integrated immune responses (antibody, CD8 þ and CD4 þ ) in nearly all vaccinated patients when given with appropriate adjuvants.

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Purpose Evaluate the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of the first-in-class investigational NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924) in patients with relapsed/refractory lymphoma or multiple myeloma. Experimental Design Patients with relapsed/refractory myeloma (n=17) or lymphoma (n=27) received intravenous pevonedistat 25–147 mg/m2 on days 1, 2, 8, 9 (schedule A; n=27) or 100–261 mg/m2 on days 1, 4, 8, 11 (schedule B; n=17) of 21-day cycles. Results Maximum tolerated doses were 110 mg/m2 (schedule A) and 196 mg/m2 (schedule B). Dose-limiting toxicities included febrile neutropenia, transaminase elevations, muscle cramps (schedule A), and thrombocytopenia (schedule B). Common adverse events included fatigue and nausea. Common grade ≥3 events were anemia (19%; schedule A), and neutropenia and pneumonia (12%; schedule B). Clinically significant myelosuppression was uncommon. There were no treatment-related deaths. Pevonedistat pharmacokinetics exhibited a biphasic disposition phase and approximate dose-proportional increases in systemic exposure. Consistent with the short mean elimination half-life of ~8.5 hours, little-to-no drug accumulation in plasma was seen after multiple dosing. Pharmacodynamic evidence of NAE inhibition included increased skin levels of CDT-1 and NRF-2 (substrates of NAE-dependent ubiquitin ligases), and increased NRF-2-regulated gene transcript levels in whole blood. Pevonedistat-NEDD8 adduct was detected in bone marrow aspirates, indicating pevonedistat target engagement in the bone marrow compartment. Three lymphoma patients had partial responses; 30 patients achieved stable disease. Conclusions Pevonedistat demonstrated anticipated pharmacodynamic effects in the clinical setting, a tolerable safety profile, and some preliminary evidence that may be suggestive of the potential for activity in relapsed/refractory lymphoma.