Faculty Bibliography

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.

4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.

OPINION STATEMENT/UNASSIGNED:Acute myeloid leukemia (AML) is the most common form of leukemia in adults, leading to the highest number of annual leukemia-associated deaths in the USA. Although most AML patients initially enter remission following induction therapy, most eventually relapse, underscoring the unmet need for more effective therapies. In recent years, novel high-throughput sequencing techniques, and mouse and human models of disease have increased our understanding of the molecular mechanisms that lead to AML. Leukemogenic mechanisms can be broadly classified into two types-cell-intrinsic and cell-extrinsic. Cell-intrinsic mechanisms include an array of genetic and epigenetic alterations that lead to dysregulated gene expression and function in hematopoietic stem/progenitor cells, leading to their increased fitness and ultimately, malignant transformation. Extrinsic mechanisms include both hematopoietic and non-hematopoietic stromal components of the leukemic microenvironment that interact with pre-leukemic and leukemic clones to promote their survival, self-renewal, and/or resistance to therapy. Through the individual and concerted action of these factors, pre-leukemic clones acquire the changes necessary for leukemic transformation. In addition, following therapy, specific leukemic clones are selected for that eventually re-initiate disease. Improving our understanding of these cell-intrinsic and cell-extrinsic mechanisms will provide novel opportunities to treat AML as well as prevent the development of disease.

Little is known regarding T cell translational regulation. We demonstrate that T follicular helper (TFH) cells use a previously unknown mechanism of selective messenger RNA (mRNA) translation for their differentiation, role in B cell maturation, and in autoimmune pathogenesis. We show that TFH cells have much higher levels of translation factor eIF4E than non-TFH CD4⁺ T cells, which is essential for translation of TFH cell fate-specification mRNAs. Genome-wide translation studies indicate that modest down-regulation of eIF4E activity by a small-molecule inhibitor or short hairpin RN impairs TFH cell development and function. In mice, down-regulation of eIF4E activity specifically reduces TFH cells among T helper subtypes, germinal centers, B cell recruitment, and antibody production. In experimental autoimmune encephalomyelitis, eIF4E activity down-regulation blocks TFH cell participation in disease pathogenesis while promoting rapid remission and spinal cord remyelination. TFH cell development and its role in autoimmune pathogenesis involve selective mRNA translation that is highly druggable.

Acute myeloid leukemia (AML) is characterized by poor clinical outcomes due to high rates of relapse following standard-of-care induction chemotherapy. While many pathogenic drivers have been described in AML, our understanding of the molecular mechanisms mediating chemotherapy resistance remains poor. Therefore, we sought to identify resistance genes to induction therapy in AML and elucidated ALOX5 as a novel mediator of resistance to anthracycline-based therapy. ALOX5 is transcriptionally upregulated in AML patient blasts in comparison to normal hematopoietic stem/progenitor cells (HSPCs) and ALOX5 mRNA, and protein expression is increased in response to induction therapy. In vitro, and in vivo genetic, and pharmacologic perturbation studies confirm that ALOX5 positively regulates the leukemogenic potential of AML LSCs, and its loss does not significantly affect the function of normal HSPCs. ALOX5 mediates resistance to daunorubicin (DNR) and promotes AML cell survival and maintenance through its leukotriene (LT) synthetic capacity, specifically via modulating the synthesis of LTB4 and its binding to LTB receptor (BLTR). Our study reveals a previously unrecognized role of LTs in AML pathogenesis and chemoresistance, whereby inhibition of ALOX5 mediated LTB4 synthesis and function could be combined with standard chemotherapy, to enhance the overall therapeutic efficacy in AML.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain human CD99 and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.

Taspase1, a highly conserved threonine protease encoded by TASP1, cleaves nuclear histone modifying factors and basal transcription regulators to orchestrate diverse transcription programs. Hereditary loss-of-function mutation of TASP1 has recently been reported in human resulting in a novel anomaly complex syndrome manifested with hematological, facial, and skeletal abnormalities. Here, we demonstrate that Taspase1-mediated cleavage of TFIIAα-β, rather than of MLL1 or MLL2, in mouse embryos is required for proper fetal liver hematopoiesis and correct segmental identities of the axial skeleton. Homozygous genetic deletion of Taspase1 (Tasp1-/-) disrupted embryonic hematopoietic stem cell self-renewal and quiescence states, and axial skeleton fates. Strikingly, mice carrying knockin non-cleavable mutations of TFIIAα-β (Gtf2a1nc/nc), a well-characterized basal transcription factor, displayed more pronounced fetal liver and axial skeleton defects than those with non-cleavable MLL1 and MLL2 (Mll1nc/nc;2nc/nc), two trithorax group (Trx-G) histone H3 trimethyl transferases. Our study offers molecular insights concerning TASP1-loss human syndrome and discovers unexpected role of TFIIAα-β cleavage in embryonic cell fate decisions.