Faculty Bibliography

Background Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer and while curable in the majority of cases, 15%-20% of children will relapse with only 50% surviving long-term. Treatment failures arise from the outgrowth of pre-existing or acquired sub-clones that are genetically or epigenetically primed to resist treatment. In addition, the bone marrow microenvironment is known to provide a protective niche. We performed the first mapping of the human B-cell ALL (B-ALL) immune bone marrow (BM) microenvironment at single cell resolution at diagnosis, remission and relapse (Witkowski, 2020). We uncovered a striking rewiring of the myeloid compartment during B-ALL progression with significant over-representation of a leukemia-associated monocyte subpopulation expressing high levels of the Macrophage Colony Stimulating Factor Receptor (M-CSFR/CSF1R). Using both peripheral blood (PB) complete blood count analysis and RNA-seq data, we demonstrated that high monocyte abundance at B-ALL diagnosis is predictive of inferior pediatric and adult overall survival in two large independent clinical cohorts. To determine the association of non-classical monocyte abundance in BM and PB with risk of relapse, we examined a cohort of clinical samples from children enrolled on Children's Oncology Group (COG) protocols. Methods Using an unmatched case-control design, we established a preliminary cohort of PB and BM samples collected at diagnosis from 24 B-ALL patients with eventual relapse and 24 patients in long-term remission. Four remission samples from an NYU Langone cohort were used to validate the expansion of this population in the presence of B-ALL. We applied a customized flow cytometry based assay to identify CD115-expressing human monocyte subsets: classical (CD45 ⁺CD56 ^-CD14 ⁺CD16 ^-), non-classical (CD45 ⁺CD56 ^-CD14 ^-CD16 ⁺), as well as B-cells (CD19, CD22, CD10) and T/NK cells (CD3, CD56). We then performed univariate and multivariable analysis of outcome (relapse versus long-term remission) compared to monocyte subset abundance, adjusting for potential confounding factors (age, gender, CNS status, NCI risk, genetic subtype, WBC at diagnosis, and end of induction minimal residual disease). Results We observed a significantly higher percentage of non-classical monocytes in the diagnostic BM from the COG cohort when compared to remission samples (COG diagnostic B-ALL BM non-classical percentage mean 52.19% vs NYU B-ALL remission BM non-classical percentage mean 1.775%, P = 0.0001). We also observed a strong correlation between the percentage of non-classical monocytes in the PB when compared to their matched BM specimens (r = 0.6, P = 0.0001). Multivariable analysis revealed a significant association between PB non-classical monocyte percentage at diagnosis and patient outcome (remission cohort non-classical monocyte percentage [mean, range]: 52.4%, 33.3-68.1%, n = 24, relapse cohort non-classical monocyte percentage: 65.9%, 56.4-84.7%, n = 24, P = 0.021). Similarly, a strong trend was observed in the BM, although it did not reach statistical significance. Flow cytometric analysis confirmed CD115 (M-CSFR/CSF1R) expression in this non-classical monocyte population, thereby validating a novel target for intervention. Conclusions These findings validate the presence of a unique monocyte subpopulation associated with childhood B-ALL and suggests that assessing this population in PB at diagnosis may be of prognostic significance. The availability of small molecule inhibitors and monoclonal antibodies targeting CSF1R-expressing monocytes may offer a novel approach to treating B-ALL. [Formula presented] Disclosures: Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Teachey: Sobi: Consultancy; NeoImmune Tech: Research Funding; Janssen: Consultancy; BEAM Therapeutics: Consultancy, Research Funding. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy.
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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.

T cells, the main effectors of the adaptive immune system, originate from thymocyte precursors in the human thymus. These immature cells follow a tediously regulated differentiation process with several critical checkpoints to ensure the generation of a diverse repertoire of fully functional, non-self T cells. In previous studies, we already uncovered part of the regulatory mechanisms that drives this differentiation process in human, which is notably different compared to mouse (De Decker et al., 2020; Dolens et al., 2020; Lavaert et al., 2020; Roels et al., 2020a; Roels et al., 2020b). However, a lot of the regulatory complexities remain unknown. By profiling additional layers of these discrete stages of thymocytes, we here aimed to generate the most complete integrative multi-omics reference map of human T cell development so far. By using a multi-omics factor analysis approach, we combined transcriptomics, epigenomics, proteomics and finally 3D genomics into one model to fully describe the T cell developmental process. We found that human T cell development is equally affected by previously unappreciated drivers, including non-coding and circular RNA, alternative splicing and DNA methylation. While some omics layers show global and genome-wide changes within developing alphabeta and gammadelta T cells, the global 3D structure remains very stable throughout all differentiation states. Nonetheless, specific alterations do occur at developmentally important genes, especially at the boundaries of active and inactive compartments. This integrative analysis aids our understanding of immune cell development both in health and disease, and reveals important aspect of lineage differentiation dynamics

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.

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.

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.

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.

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.