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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN ® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy derived from the precursors of plasmacytoid dendritic cells. Here the authors characterize the anti-tumor activity of allogeneic anti-CD123 CAR-T cells in preclinical models of BPDCN.

The prevalence of testicular germ cell tumors (TGCT), a common solid tissue malignancy in young men, has been annually increasing at an alarming rate of 3%. Since the majority of testicular cancers are derived from germ cells at the stage of transformation of primordial germ cell (PGC) into gonocytes, the increase has been attributed to maternal/fetal exposures to environmental factors. We examined the effects of an estrogen (diethylstilbestrol, DES), an antiandrogen (flutamide), or radiation on the incidence of testicular germ cell tumors in genetically predisposed 129.MOLF-L1 (L1) congenic mice by exposing them to these agents on days 10.5 and 11.5 of pregnancy. Neither flutamide nor DES produced noticeable increases in testis cancer incidence at 4 weeks of age. In contrast, two doses of 0.8-Gy radiation increased the incidence of TGCT from 45% to 100% in the offspring. The percentage of mice with bilateral tumors, weights of testes with TGCT, and the percentage of tumors that were clearly teratomas were higher in the irradiated mice than in controls, indicating that irradiation induced more aggressive tumors and/or more foci of initiation sites in each testis. This radiation dose did not disrupt spermatogenesis, which was qualitatively normal in tumor-free testes although they were reduced in size. This is the first proof of induction of testicular cancer by an environmental agent and suggests that the male fetus of women exposed to radiation at about 5-6 weeks of pregnancy might have an increased risk of developing testicular cancer. Furthermore, it provides a novel tool for studying the molecular and cellular events of testicular cancer pathogenesis.

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2–selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

BackgroundAcute myeloid leukemia (AML) stem cells (LSCs) are capable of surviving current standard chemotherapy and are the likely source of deadly, relapsed disease. While stem cell transplant serves as proof-of-principle that AML LSCs can be eliminated by the immune system, the translation of existing immunotherapies to AML has been met with limited success. Consequently, understanding and exploiting the unique immune-evasive mechanisms of AML LSCs is critical.MethodsAnalysis of stem cell datasets and primary patient samples revealed CD200 as a putative stem cell–specific immune checkpoint overexpressed in AML LSCs. Isogenic cell line models of CD200 expression were employed to characterize the interaction of CD200+ AML with various immune cell subsets both in vitro and in peripheral blood mononuclear cell (PBMC)–humanized mouse models. CyTOF and RNA-sequencing were performed on humanized mice to identify novel mechanisms of CD200-mediated immunosuppression. To clinically translate these findings, we developed a fully humanized CD200 antibody (IgG1) that removed the immunosuppressive signal by blocking interaction with the CD200 receptor while also inducing a potent Fc-mediated response. Therapeutic efficacy of the CD200 antibody was evaluated using both humanized mice and patient-derived xenograft models.ResultsOur results demonstrate that CD200 is selectively overexpressed in AML LSCs and is broadly immunosuppressive by impairing cytokine secretion in both innate and adaptive immune cell subsets. In a PBMC-humanized mouse model, CD200+ leukemia progressed rapidly, escaping elimination by T cells, compared with CD200− AML. T cells from mice with CD200+ AML were characterized by an abundance of metabolically quiescent CD8+ central and effector memory cells. Mechanistically, CD200 expression on AML cells significantly impaired OXPHOS metabolic activity in T cells from healthy donors. Importantly, CD200 antibody therapy could eliminate disease in the presence of graft-versus-leukemia in immune competent mice and could significantly improve the efficacy of low-intensity azacitidine/venetoclax chemotherapy in immunodeficient hosts.ConclusionsOverexpression of CD200 is a stem cell–specific marker that contributes to immunosuppression in AML by impairing effector cell metabolism and function. CD200 antibody therapy is capable of simultaneously reducing CD200-mediated suppression while also engaging macrophage activity. This study lays the groundwork for CD200-targeted therapeutic strategies to eliminate LSCs and prevent AML relapse.

Irradiation causes spermatogenic arrest in adult rats and this arrest can be reversed by reduction of intratesticular testosterone by GnRH-antagonist, whereas further testosterone treatment attenuates the spermatogenesis recovery. To identify hormonally regulated genes correlating with the spermatogenic differentiation status, gene expression profiles were investigated by microarray with Rat 230 2.0 GeneChips (Affymetrix), comparing normal control rats, irradiated rats with no treatment, irradiated rats treated with GnRH-antagonist and either flutamide (androgen receptor antagonist) or testosterone, for 2 weeks, at which time there were minimal changes in number of germ cells. Insulin-like 3 (Insl3), a member of the relaxin-insulin hormone family was the second most strongly down-regulated gene by testosterone suppression. Real-time RT-PCR confirmed microarray observations: after irradiation, relative Insl3 mRNA level increased about 9-fold above the normal control; suppression of testosterone by GnRH-antagonist and flutamide repressed Insl3 level by 20-fold compared to the irradiated-only group; adding testosterone together with GnRH antagonist elevated Insl3 level so that it was only 6-fold reduced from the irradiated only group. Characterization of the changes in INSL3 protein levels by immunoblot supported the mRNA results: irradiation caused over 10-fold increase in relative prepro-(or pro-)INSL3 protein levels compared to that of normal controls; treatment of GnRH-antagonist and flutamide down-regulated INSL3 protein to an undetectable level; supplementation with testosterone restored INSL3 protein levels that were 7-fold reduced from the irradiated-only group. As a first step to determining if the down-regulation of Insl3 mRNA level by androgen suppression is direct or indirect, irradiated rats were treated with GnRH-antagonist + flutamide for 1, 3, and 7 days, respectively. One-day androgen suppression induced only a 1.3-fold reduction in Insl3 mRNA level compared to irradiated-only rats; 3 days of androgen suppression resulted in 4-fold Insl3 mRNA reduction; and 7 days androgen depletion caused a 10-fold reduction in Insl3 mRNA level. Meanwhile, one day GnRH-antagonist + flutamide reduced INSL3 protein 1.7-fold, and both 3 days and 7 days androgen depletion suppressed INSL3 protein to undetectable levels. Measurement of intratesticular testosterone levels will give us more insight on whether Insl3 is directly or indirectly regulated by androgen. Similar regulation of Insl3 mRNA by androgen has been observed in germ cell depleted (e.g. jsd) mice treated with GnRH-antagonist plus flutamide or testosterone. Thus INSL3 levels are associated with spermatogenesis arrest/recovery in at least two different pathogenic models. Although INSL3 plays a major role in testicular descent during embryonic development by acting on the gubernaculum, little is known about its function in the adult. Further characterization of INSL3 regulation will help us understand the role of INSL3 in the recovery of spermatogenesis after toxicant treatment of adult mammals. (Supported by Grant ES-08075 from NIEHS and HD-40397 from NICHD)

Overexpression of REST has been implicated in brain tumors, ischemic insults, epilepsy, and movement disorders such as Huntington’s disease. However, owing to the lack of a conditional REST overexpression animal model, the mechanism of action of REST overexpression in these disorders has not been established in vivo . We created a REST overexpression mouse model using the human REST ( hREST ) gene. Our results using these mice confirm that hREST expression parallels endogenous REST expression in embryonic mouse brains. Further analyses indicate that REST represses the dopamine receptor 2 ( Drd2 ) gene, which encodes a critical nigrostriatal receptor involved in regulating movement, in vivo . Overexpression of REST using Drd2-Cre in adult mice results in increased REST and decreased DRD2 expression in the striatum, a major site of DRD2 expression, and phenocopies the spontaneous locomotion deficits seen upon global DRD2 deletion or specific DRD2 deletion from indirect-pathway medium spiny neurons. Thus, our studies using this mouse model not only reveal a new function of REST in regulating spontaneous locomotion but also suggest that REST overexpression in DRD2-expressing cells results in spontaneous locomotion deficits.