Search

Survival of infants with KMT2A-rearranged acute lymphoblastic leukemia (ALL) remains dismal despite intensive chemotherapy. We observed constitutive phosphorylation of spleen tyrosine kinase (SYK) and associated signaling proteins in infant ALL patient-derived xenograft (PDX) model specimens and hypothesized that the SYK inhibitor entospletinib would inhibit signaling and cell growth in vitro and leukemia proliferation in vivo. We further predicted that combined entospletinib and chemotherapy could augment anti-leukemia effects. Basal kinase signaling activation and HOXA9/MEIS1 expression differed among KMT2Arearranged (KMT2A-AFF1 [n=4], KMT2A-MLLT3 [n=1], KMT2A-MLLT1 [n=4]) and non-KMT2A-rearranged [n=3] ALL specimens and stratified by genetic subgroup. Incubation of KMT2A-rearranged ALL cells in vitro with entospletinib inhibited methylcellulose colony formation and SYK pathway signaling in a dose-dependent manner. In vivo inhibition of leukemia proliferation with entospletinib monotherapy was observed in RAS-wild-type KMT2A-AFF1, KMT2A-MLLT3, and KMT2A-MLLT1 ALL PDX models with enhanced activity in combination with vincristine chemotherapy in several models. Surprisingly, entospletinib did not decrease leukemia burden in two KMT2A-AFF1 PDX models with NRAS or KRAS mutations, suggesting potential RAS-mediated resistance to SYK inhibition. As hypothesized, superior inhibition of ALL proliferation was observed in KMT2A-AFF1 PDX models treated with entospletinib and the MEK inhibitor selumetinib versus vehicle or inhibitor monotherapies (P

BackgroundSuccessful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown.MethodsIn this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML.ResultsWe observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A ‘best-in-class’ lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation.ConclusionsCD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML.Trial registration numberclinicaltrials.gov;NCT03971799.

Thrombocytopenia is a major complication in hematopoietic-acute radiation syndrome (H-ARS) that increases the risk of mortality from uncontrolled hemorrhage. There is a great demand for new therapies to improve survival and mitigate bleeding in H-ARS. Thrombopoiesis requires interactions between megakaryocytes (MKs) and endothelial cells. 16, 16-dimethyl prostaglandin E(2) (dmPGE(2)), a longer-acting analogue of PGE(2), promotes hematopoietic recovery after total-body irradiation (TBI), and various angiotensin-converting enzyme (ACE) inhibitors mitigate endothelial injury after radiation exposure. Here, we tested a combination therapy of dmPGE(2) and lisinopril to mitigate thrombocytopenia in murine models of H-ARS following TBI. After 7.75 Gy TBI, dmPGE(2) and lisinopril each increased survival relative to vehicle controls. Importantly, combined dmPGE(2) and lisinopril therapy enhanced survival greater than either individual agent. Studies performed after 4 Gy TBI revealed reduced numbers of marrow MKs and circulating platelets. In addition, sublethal TBI induced abnormalities both in MK maturation and in in vitro and in vivo platelet function. dmPGE(2), alone and in combination with lisinopril, improved recovery of marrow MKs and peripheral platelets. Finally, sublethal TBI transiently reduced the number of marrow Lin(−)CD45(−)CD31(+)Sca-1(−) sinusoidal endothelial cells, while combined dmPGE(2) and lisinopril treatment, but not single-agent treatment, accelerated their recovery. Taken together, these data support the concept that combined dmPGE(2) and lisinopril therapy improves thrombocytopenia and survival by promoting recovery of the MK lineage, as well as the MK niche, in the setting of H-ARS.

In light of the indispensible role of platelets in the maintenance of hemostasis, understanding the biology of platelet production from bone marrow megakaryocytes (MKs) may uncover new therapeutic strategies for thrombocytopenia. While there has been much recent interest in optimizing culture systems to facilitate the study of the morphologically unique MK lineage, these systems lack the intricacy of in vivo megakaryopoiesis. Given the limitations of many common techniques for the in vivo study of MKs, in this chapter we describe a method to quantify and analyze primary murine bone marrow megakaryocytes utilizing imaging flow cytometry.

Life-threatening thrombocytopenia can develop following bone marrow injury due to decreased platelet production from megakaryocytes (MKs). However, the study of primary MKs has been complicated by their low frequency in the bone marrow and by technical challenges presented by their unique maturation properties. More accurate and efficient methods for the analysis of in vivo MKs are needed to enhance our understanding of megakaryopoiesis and ultimately develop new therapeutic strategies for thrombocytopenia. Imaging flow cytometry (IFC) combines the morphometric capabilities of microscopy with the high-throughput analyses of flow cytometry (FC). Here, we investigate the application of IFC on the ImageStream(X) platform to the analysis of primary MKs isolated from murine bone marrow. Our data highlight and address technical challenges for conventional FC posed by the wide range of cellular size within the MK lineage as well as the shared surface phenotype with abundant platelet progeny. We further demonstrate that IFC can be used to reproducibly and efficiently quantify the frequency of primary murine MKs in the marrow, both at steady-state and in the setting of radiation-induced bone marrow injury, as well as assess their ploidy distribution. The ability to accurately analyze the full spectrum of maturing MKs in the bone marrow now allows for many possible applications of IFC to enhance our understanding of megakaryopoiesis and platelet production.

Previous work has demonstrated that cross talk between G protein-coupled LH receptors and epidermal growth factor receptors (EGFR) is essential for LH-induced steroid production in ovarian follicles and testicular Leydig cells. Here we demonstrate that G protein-coupled receptor (GPCR)/EGFR cross talk is also required for ACTH-induced steroidogenesis in Y1 adrenal cells. Moreover, we confirm that the signaling pathway from GPCR to Erk activation is conserved in all three steroidogenic tissues. ACTH or LH induces Gα(s), resulting in elevated cAMP and protein kinase A activation. cAMP/protein kinase A then triggers EGFR trans-activation, which promotes Erk signaling and subsequent steroidogenesis. Interestingly, although EGFR trans-activation is conserved in all three tissues, the specific mechanisms regulating this receptor cross talk differ. ACTH and LH trigger matrix metalloproteinase (MMP)-mediated release of EGFR ligands in adrenal and gonadal cells, respectively. However, this extracellular, ligand-dependent EGFR transactivation is required only for LH-induced steroidogenesis in ovarian follicles, reflecting the unique requirement of cell-cell cross talk for ovarian steroid production. Furthermore, MMP2 and MMP9 appear to regulate LH-induced steroidogenesis in mouse ovarian follicles, because a specific MMP2/9 inhibitor as well as the MMP2/9 inhibitor doxycycline suppress LH-induced follicular steroid production in vitro. Notably, although EGFR or MMP inhibition minimally affects estrous cycling in female mice, they attenuate ovarian steroidogenesis in response to LHR overstimulation in vivo. These results may have implications with regard to EGFR inhibitor use in various cancers as well as in polycystic ovarian syndrome, where excess LH-driven ovarian androgen production might be controlled by MMP2/9 inhibition.

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5′ splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I., Author Summary Human infertility and reproductive complications have devastating social and monetary costs. Errors in meiosis during reproduction may lead to birth defects, spontaneous abortion, or infertility. Many of the genes essential for meiosis function in DNA repair and mutations in several of these genes have been shown to contribute to cancer. The identification of the genes necessary for normal meiosis is an important goal and will potentially influence the fields of reproductive and cancer biology. In this study, genetic screens in mice have generated the mutation mei4. mei4 causes male and female sterility by disrupting meiosis and altering the function of the DNA repair system known as mismatch repair. We have identified the causative mutation behind the mei4 phenotype in a gene called Human Enhancer of Invasion 10 or Hei10. This work demonstrates that Hei10 is essential for the completion of meiosis and that it functions to coordinate the DNA repair system and the progression of the cell cycle during meiosis.

Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1β accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin., Author Summary Genes related to the sexual regulator Doublesex of Drosophila have been found to control sexual development in a wide variety of animals, ranging from roundworms to mammals. In this paper, we investigate the function of the Dmrt7 gene, one of seven related genes in the mouse. Female mammals are XX and males are XY, a chromosomal difference that presents specific challenges during the meiotic phase of male germ cell development. Some of these are thought to be overcome by incorporating the X and Y chromosomes into a specialized structure called the XY body. We find that DMRT7 protein is present in germ cells, localizes to the male XY body during meiosis, and is essential for male but not female fertility. The XY body normally is altered by recruitment of additional proteins and by specific modifications to histone proteins between the pachytene and diplotene stages of meiosis, but modification of the “sex chromatin” in Dmrt7 mutant cells is abnormal during this period. Because Dmrt7 is found in all branches of mammals, but not in other vertebrates, these results may indicate some commonality in regulation of sex chromatin among the mammals.