Faculty Bibliography

Multiple myeloma (MM) is a plasma cell malignancy characterized by the presence of multiple foci in the skeleton. These distinct tumor foci represent cycles of tumor growth and dissemination that seed new clusters and drive disease progression. By using an intratibial Vk*MYC murine myeloma model, we found that CD169+ radiation-resistant tissue-resident macrophages (MPs) were critical for early dissemination of myeloma and disease progression. Depletion of these MPs had no effect on tumor proliferation, but it did reduce egress of myeloma from bone marrow (BM) and its spread to other bones. Depletion of MPs as a single therapy and in combination with BM transplantation improved overall survival. Dissemination of myeloma was correlated with an increased inflammatory signature in BM MPs. It was also correlated with the production of interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) by tumor-associated MPs. Exogenous intravenous IL-6 and TNFα can trigger myeloma intravasation in the BM by increasing vascular permeability in the BM and by enhancing the motility of myeloma cells by reducing the adhesion of CD138. Moreover, mice that lacked IL-6 had defects in disseminating myeloma similar to those in MP-depleted recipients. Mice that were deficient in TNFα or TNFα receptor (TNFR) had defects in disseminating MM, and engraftment was also impaired. These effects on dissemination of myeloma required production of cytokines in the radiation-resistant compartment that contained these radiation-resistant BM MPs. Taken together, we propose that egress of myeloma cells from BM is regulated by localized inflammation in foci, driven in part by CD169+ MPs.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, created an unprecedented need for comprehensive laboratory testing of populations, in order to meet the needs of medical practice and to guide the management and functioning of our society. With the greater New York metropolitan area as an epicenter of this pandemic beginning in March 2020, a consortium of laboratory leaders from the assembled New York academic medical institutions was formed to help identify and solve the challenges of deploying testing. This report brings forward the experience of this consortium, based on the real-world challenges which we encountered in testing patients and in supporting the recovery effort to reestablish the health care workplace. In coordination with the Greater New York Hospital Association and with the public health laboratory of New York State, this consortium communicated with state leadership to help inform public decision-making addressing the crisis. Through the length of the pandemic, the consortium has been a critical mechanism for sharing experience and best practices in dealing with issues including the following: instrument platforms, sample sources, test performance, pre- and post-analytical issues, supply chain, institutional testing capacity, pooled testing, biospecimen science, and research. The consortium also has been a mechanism for staying abreast of state and municipal policies and initiatives, and their impact on institutional and laboratory operations. The experience of this consortium may be of value to current and future laboratory professionals and policy-makers alike, in dealing with major events that impact regional laboratory services.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.

Lack of cellular differentiation is a hallmark of many human cancers, including acute myeloid leukemia (AML). Strategies to overcome such a differentiation blockade are an approach for treating AML. To identify targets for differentiation-based therapies, we applied an integrated cell surface-based CRISPR platform to assess genes involved in maintaining the undifferentiated state of leukemia cells. Here we identify the RNA-binding protein ZFP36L2 as a critical regulator of AML maintenance and differentiation. Mechanistically, ZFP36L2 interacts with the 3' untranslated region of key myeloid maturation genes, including the ZFP36 paralogs, to promote their mRNA degradation and suppress terminal myeloid cell differentiation. Genetic inhibition of ZFP36L2 restores the mRNA stability of these targeted transcripts and ultimately triggers myeloid differentiation in leukemia cells. Epigenome profiling of several individuals with primary AML revealed enhancer modules near ZFP36L2 that associated with distinct AML cell states, establishing a coordinated epigenetic and post-transcriptional mechanism that shapes leukemic differentiation.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4⁺CD8⁺ T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL.

Mechanical loading (ML) is a potent anabolic stimulus in healthy adult bone [1]. A better understanding of cell and molecular processes in load-induced osteogenesis, including underlying mechanosensitive pathways, could yield effective treatment strategies for aged and diseased bone. Cortical bone (CB) osteocytes (OCYs) originate from LepR+ cells, of which 98.8% co-express CXCL12 [2]. Given that skeletal stem cells (SSCs) express an array of overlapping markers, including LepR and CXCL12 [2-6], we sought to determine how these distinct SSC populations respond to ML with regard to their number and gene expression profiles at the single cell level. We hypothesized that ML leads to an expansion of CXCL12+ and LepR+ cell populations and regulates their fate. Following NYU IACUC approval, adult Cxcl12tm2.1Sjm/J dsRed reporter mice (N=19) and C57BL/6 (C57, N=6) mice (Jackson Labs) were subjected to 4 daily bouts of tibial axial compressive loading (L) (6N,2Hz,120cycles) with appropriate non-loaded (NL) controls. Bone marrow (BM) and CB cell suspensions from L and NL tibiae were prepared for FACS and single-cell RNAseq (10XGenomics) as previously described [5]. FACS data are presented as %change and significance determined by a Student's T-test at alpha=0.05; expression data are presented as normalized fold-change. The enriched CXCL12+ cell population was shown to highly express LepR. ML led to a significant increase in the number of LepR+ cells (+114%, p=0.022) in the BM. Differentially expressed genes in the L versus NL CXCL12-dsRed+ cells included upregulated osteogenic genes Wnt4 (1.32X, p<0.0001) and BMP4 (1.72X, p<0.0001), and downregulated adipo-associated genes PPARgamma (0.79X, p=0.037) and Apoe (0.92X, p<0.001). Unbiased principle component analysis (PCA) yielded 11 cell clusters, including reticular cells [2,5,6] and pre-osteoblasts [5,6]. Loading resulted in a significant increase in BMP4 (2.7X, p=0.002) and a significant decrease in sFRP expression (0.55X, p<0.0001), 10 a negative regulator of Wnt signaling [7], in reticular cells. A significant increase in Wnt4 (1.9X, p<0.0001) was also observed in pre-osteoblasts. Our data demonstrate that loading promotes osteogenic differentiation via promotion of Wnt signaling, consistent with previous reports [8-10] while attenuating pro-adipogenic genes in cells expressing both LepR and CXCL12, which are known osteoprogenitors [2]; that is, loading effectively pushes progenitors towards an osteogenic fate

B cell acute lymphoblastic leukemia (B-ALL) blasts hijack the bone marrow (BM) microenvironment to form chemoprotective leukemic BM "niches," facilitating chemoresistance and, ultimately, disease relapse. However, the ability to dissect these evolving, heterogeneous interactions among distinct B-ALL subtypes and their varying BM niches is limited with current in vivo methods. Here, we demonstrated an in vitro organotypic "leukemia-on-a-chip" model to emulate the in vivo B-ALL BM pathology and comparatively studied the spatial and genetic heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. We revealed the heterogeneous chemoresistance mechanisms across various B-ALL cell lines and patient-derived samples. We showed that the leukemic perivascular, endosteal, and hematopoietic niche-derived factors maintain B-ALL survival and quiescence (e.g., CXCL12 cytokine signal, VCAM-1/OPN adhesive signals, and enhanced downstream leukemia-intrinsic NF-κB pathway). Furthermore, we demonstrated the preclinical use of our model to test niche-cotargeting regimens, which may translate to patient-specific therapy screening and response prediction.