Faculty Bibliography

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.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intestinal crypts have great capacity for repair and regeneration after intestinal stem cell (ISC) injury. Here, we define the cellular remodeling process resulting from ISC niche interruption by transient Notch pathway inhibition in adult mice. Although ISCs were retained, lineage tracing demonstrated a marked reduction in ISC function after Notch disruption. Surprisingly, Notch ligand-expressing Paneth cells were rapidly lost by apoptotic cell death. The ISC-Paneth cell changes were followed by a regenerative response, characterized by expansion of cells expressing Notch ligands Dll1 and Dll4, enhanced Notch signaling, and a proliferative surge. Lineage tracing and organoid studies showed that Dll1-expressing cells were activated to function as multipotential progenitors, generating both absorptive and secretory cells and replenishing the vacant Paneth cell pool. Our analysis uncovered a dynamic, multicellular remodeling response to acute Notch inhibition to repair the niche and restore homeostasis. Notably, this crypt regenerative response did not require ISC loss.