FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Inhibiting the biogenesis of myeloid-derived suppressor cells enhances immunotherapy efficacy against mammary tumor progression.

While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1-based (PD-1-based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8+ T cell responses. Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.

Indoleamine 2,3-dioxygenase 1 is essential for sustaining durable antibody responses.

Humoral immunity is essential for protection against pathogens, emphasized by the prevention of 2-3 million deaths worldwide annually by childhood immunizations. Long-term protective immunity is dependent on the continual production of neutralizing antibodies by the subset of long-lived plasma cells (LLPCs). LLPCs are not intrinsically long-lived, but require interaction with LLPC niche stromal cells for survival. However, it remains unclear which and how these interactions sustain LLPC survival and long-term humoral immunity. We now have found that the immunosuppressive enzyme indoleamine 2,3- dioxygenase 1 (IDO1) is required to sustain antibody responses and LLPC survival. Activation of IDO1 occurs upon the engagement of CD80/CD86 on the niche dendritic cells by CD28 on LLPC. Kynurenine, the product of IDO1 catabolism, activates the aryl hydrocarbon receptor in LLPC, reinforcing CD28 expression and survival signaling. These findings expand the immune function of IDO1 and uncover a novel pathway for sustaining LLPC survival and humoral immunity.

Inhibition of LSD1 in MDS progenitors restores differentiation of CD141Hi conventional dendritic cells.

The use of immunotherapy to treat patients with myelodysplastic syndromes (MDS) shows promise but is limited by our incomplete understanding of the immunologic milieu. In solid tumors, CD141Hi conventional dendritic cells (CD141Hi cDCs) are necessary for antitumor immunosurveillance and the response to immunotherapy. Here, we found that CD141Hi cDCs are reduced in MDS bone marrow and based on the premise established in solid tumors, we hypothesized that reduced numbers of CD141Hi cDCs are associated with inferior overall survival in MDS patients. We found that MDS patients with reduced numbers of CD141Hi cDCs, but not other DC populations, showed reduced overall survival. To examine the basis for reduction in CD141Hi cDCs, we found fewer numbers of progenitors committed to DC differentiation in the MDS bone marrow and these progenitors expressed lower levels of interferon regulatory factor-8 (IRF8), a master regulator of CD141Hi cDC differentiation. To rescue impaired CD141Hi cDC differentiation, we used pharmacologic inhibition of lysine-specific demethylase 1A (LSD1) to promote CD141Hi cDC differentiation by MDS progenitors. These data reveal a previously unrecognized element of the MDS immunologic milieu. Epigenetic regulation of CD141Hi cDC differentiation offers an intriguing opportunity for intervention and a potential adjunct to immunotherapy for patients with MDS.

SSeCKS/Akap12 suppresses metastatic melanoma lung colonization by attenuating Src-mediated pre-metastatic niche crosstalk.

SSeCKS/Gravin/AKAP12 (SSeCKS) controls metastasis-associated PKC and Src signaling through direct scaffolding activity. SSeCKS is downregulated in the metastases of many human cancer types, and its forced re-expression suppresses the metastatic behavior of prostate cancer cells. SSeCKS is also downregulated in breast and prostate cancer stroma, and SSeCKS-null mice (KO) are metastasis-prone, suggesting a role in suppressing formation of the pre-metastatic niche. Here, we show that lung colonization and metastasis formation by B16F10 and SM1WT1[Braf V600E] mouse melanoma cells is 9-fold higher in syngeneic KO compared to WT hosts, although there is no difference in orthotopic tumor volumes. Although melanoma cells adhered equally to KO or WT lung fibroblasts (LF), co-injection of melanoma cells with KO (vs. WT) LF increased lung macrometastasis formation in WT hosts, marked by increased melanoma colonization at foci of leaky vasculature. Increased melanoma adhesion on KO lung endothelial cells (LEC) was facilitated by increased E-Selectin levels and by increased STAT3-regulated secretion of senescence-associated factors from KO-LF, such as Vegf. Finally, the ability of SSeCKS to attenuate IFNα-induced Stat3 activation in KO-LF required its Src-scaffolding domain. Taken together, these data suggest that SSeCKS normally suppresses metastatic colonization in the lung by attenuating the expression of Selectin adhesion proteins, which can be controlled autonomously by local endothelial cells or enhanced by senescence factors secreted by neighboring fibroblasts in a SSeCKS-regulated, Src/Stat3-dependent manner.

SSeCKS/AKAP12 scaffolding functions suppress B16F10-induced peritoneal metastasis by attenuating CXCL9/10 secretion by resident fibroblasts.

SSeCKS/Gravin/AKAP12 (SSeCKS) is a kinase scaffolding protein known to suppress metastasis by attenuating tumor-intrinsic PKC- and Src-mediated signaling pathways [1]. In addition to downregulation in metastatic cells, in silico analyses identified SSeCKS downregulation in prostate or breast cancer-derived stroma, suggesting a microenvironmental cell role in controlling malignancy. Although orthotopic B16F10 and SM1WT1[BrafV600E] mouse melanoma tumors grew similarly in syngeneic WT or SSeCKS-null (KO) mice, KO hosts exhibited 5- to 10-fold higher levels of peritoneal metastasis, and this enhancement could be adoptively transferred by pre-injecting naïve WT mice with peritoneal fluid (PF), but not non-adherent peritoneal cells (PC), from naïve KO mice. B16F10 and SM1WT1 cells showed increased chemotaxis to KO-PF compared to WT-PF, corresponding to increased PF levels of multiple inflammatory mediators, including the Cxcr3 ligands, Cxcl9 and 10. Cxcr3 knockdown abrogated enhanced chemotaxis to KO-PF and peritoneal metastasis in KO hosts. Conditioned media from KO peritoneal membrane fibroblasts (PMF), but not from KO-PC, induced increased B16F10 chemotaxis over controls, which could be blocked with Cxcl10 neutralizing antibody. KO-PMF exhibited increased levels of the senescence markers, SA-ß-galactosidase, p21waf1 and p16ink4a, and enhanced Cxcl10 secretion induced by inflammatory mediators, lipopolysaccharide, TNFα, IFNα and IFNγ. SSeCKS scaffolding-site mutants and small molecule kinase inhibitors were used to show that the loss of SSeCKS-regulated PKC, PKA and PI3K/Akt pathways are responsible for the enhanced Cxcl10 secretion. These data mark the first description of a role for stromal SSeCKS/AKAP12 in suppressing metastasis, specifically by attenuating signaling pathways that promote secretion of tumor chemoattractants in the peritoneum.

Genetic dissection of the Down syndrome critical region.

Down syndrome (DS), caused by trisomy 21, is the most common chromosomal disorder associated with developmental cognitive deficits. Despite intensive efforts, the genetic mechanisms underlying developmental cognitive deficits remain poorly understood, and no treatment has been proven effective. The previous mouse-based experiments suggest that the so-called Down syndrome critical region of human chromosome 21 is an important region for this phenotype, which is demarcated by Setd4/Cbr1 and Fam3b/Mx2. We first confirmed the importance of the Cbr1-Fam3b region using compound mutant mice, which carry a duplication spanning the entire human chromosome 21 orthologous region on mouse chromosome 16 [Dp(16)1Yey] and Ms1Rhr. By dividing the Setd4-Mx2 region into complementary Setd4-Kcnj6 and Kcnj15-Mx2 intervals, we started an unbiased dissection through generating and analyzing Dp(16)1Yey/Df(16Setd4-Kcnj6)Yey and Dp(16)1Yey/Df(16Kcnj15-Mx2)Yey mice. Surprisingly, the Dp(16)1Yey-associated cognitive phenotypes were not rescued by either deletion in the compound mutants, suggesting the possible presence of at least one causative gene in each of the two regions. The partial rescue by a Dyrk1a mutation in a compound mutant carrying Dp(16)1Yey and the Dyrk1a mutation confirmed the causative role of Dyrk1a, whereas the absence of a similar rescue by Df(16Dyrk1a-Kcnj6)Yey in Dp(16)1Yey/Df(16Dyrk1a-Kcnj6)Yey mice demonstrated the importance of Kcnj6. Our results revealed the high levels of complexities of gene actions and interactions associated with the Setd4/Cbr1-Fam3b/Mx2 region as well as their relationship with developmental cognitive deficits in DS.

Differential requirement for Src family tyrosine kinases in the initiation, progression, and metastasis of prostate cancer.

UNLABELLED: Prostate cancer (CaP) recurrence after androgen ablation therapy remains a significant cause of mortality in aging men. Malignant progression and metastasis are typically driven by genetic and epigenetic changes controlled by the androgen receptor (AR). However, evidence suggests that activated nonreceptor tyrosine kinases, including those of the Src family kinases (SFK), directly phosphorylate AR, thereby activating its transcriptional activity in the absence of serum androgen levels. To ascertain whether CaP progression and metastasis require SFK members, an autochthonous transgenic adenocarcinoma (AD) of the mouse prostate (TRAMP) model was crossed into Src-, Lyn- or Fyn-null backgrounds. Primary-site CaP formation was dependent on Src, to a lesser extent, Lyn, but not Fyn. Only Src(-) (/) (-);TRAMP prostate tumors were marked by reactive stroma. SFK deficiency did not affect progression to neuroendocrine (NE) disease, although there were fewer new cancer cases initiating after 34 weeks in the SFK(-/-);TRAMP mice compared with TRAMP controls. Of note, 15% to 21% of older (>33 weeks) Lyn- or Fyn-null TRAMP mice lacking primary-site tumors suffered from aggressive metastatic AD growths, compared with 3% of TRAMP mice. Taken with the data that TRAMP mice lacking Src or Lyn exhibited fewer macroscopic metastases compared with Fyn(-) (/) (-);TRAMP and TRAMP controls, this suggests that SFK can either promote or suppress specific parameters of metastatic growth, possibly depending on cross-talk with primary tumors. These data identify critical, yet potentially opposing roles played by various SFKs in the initiation and metastatic potential of CaP using the TRAMP model. IMPLICATIONS: Genetically defined mouse models indicate a critical role for Src tyrosine kinase in CaP initiation and metastatic progression.

A transgenic mouse model for early prostate metastasis to lymph nodes.

The emergence of recurrent, metastatic prostate cancer following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, little is known regarding the genes and pathways that regulate this metastatic process, and moreover, it is unclear whether metastasis is an early or late event. The individual genetic loss of the metastasis suppressor, SSeCKS/Gravin/AKAP12 or Rb, genes that are downregulated or deleted in human prostate cancer, results in prostatic hyperplasia. Here, we show that the combined loss of Akap12 and Rb results in prostatic intraepithelial neoplasia (PIN) that fails to progress to malignancy after 18 months. Strikingly, 83% of mice with PIN lesions exhibited metastases to draining lymph nodes, marked by relatively differentiated tumor cells expressing markers of basal (p63, cytokeratin 14) and luminal (cytokeratin 8 and androgen receptor) epithelial cells, although none expressed the basal marker, cytokeratin 5. The finding that PIN lesions contain increased numbers of p63/AR-positive, cytokeratin 5-negative basal cells compared with WT or Akap12-/- prostate lobes suggests that these transitional cells may be the source of the lymph node metastases. Taken together, these data suggest that in the context of Rb loss, Akap12 suppresses the oncogenic proliferation and early metastatic spread of basal-luminal prostate tumor cells.

Gonadotropin stimulation as a challenge to calibrate cisplatin induced ovarian damage in the female rat.

Cisplatin administration for treatment of cancer causes damage to the ovaries, potentially leading to ovarian failure. To extend our previous work, we tested the hypothesis that serum anti-Mullerian hormone (AMH) levels after ovarian stimulation can be used as a biomarker to calibrate the degree of ovarian damage following cisplatin. Female rats were injected with two weekly doses of cisplatin followed by an injection of pregnant mare serum gonadotropin to stimulate the ovaries. Sera and ovaries were collected 54 h after gonadotropin challenge. Analysis of serum and ovarian AMH by Western blot and ELISA revealed that increasing doses of cisplatin caused dose-dependent decrease in AMH after gonadotropin stimulation. Immunohistochemical analysis demonstrated that the AMH positive follicles declined in a dose-dependent manner after cisplatin, resulting in AMH levels that are lower after cisplatin exposure. Cisplatin damage to the ovaries can be identified by the serum AMH levels following exogenous ovarian stimulation.

Declines in levels of hyperpolarization-activated cation (HCN) channels in the rat ovary after cisplatin exposure.

The distribution of the 4 hyperpolarization-activated cation (hyperpolarization-activated, cyclic nucleotide-gated [HCN]) channels (HCN1-4), channels associated with rhythmic electrical bursting, varies in the different cells of the ovary. Cisplatin is a broadly used chemotherapeutic agent that results in ovarian damage. The objective of this study was to test the hypothesis that cisplatin treatment will affect expression of the ovarian HCN channels. Adult female Sprague-Dawley rats were dosed with saline, 4.5 mg/kg cisplatin, or 6.0 mg/kg cisplatin as 2 weekly injections and then were killed 5 days after the second cisplatin dose. The ovaries were studied for HCN1-4 using semiquantitative H score immunohistochemical analysis and using Western blot analysis. By immunohistochemistry, for HCN4, the oocyte-specific staining declined after cisplatin treatment (P < .001). There was a decline in HCN2 intensity after cisplatin in the antral granulosa cells (P < .05) and thecal cells (P < .05). There were no differences found for the immunohistochemical HCN1 and HCN3 H scores after cisplatin. The Western blot analysis revealed that there was a decline in HCN3 levels after cisplatin exposure (P < .05). For HCN1 and HCN2, there was no change in the total ovarian protein levels after cisplatin. Treatment in vivo with cisplatin resulted in decline in the levels of the HCN channels in the rat ovary, with oocytes, antral cells, and thecal cells being specifically affected.

The expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation.

BACKGROUND: Aim of this study was to test the hypothesis that levels of hyperpolarization activated cyclic nucleotide gated channels 1 to 4 (HCN1-4) are linked to the reproductive age of the ovary. METHODS: Young, adult, and reproductively aged ovaries were collected from Sprague-Dawley rats. RT-PCR and western blot analysis of ovaries was performed to investigate the presence of mRNA and total protein for HCN1-4. Immunohistochemistry with semiquantitative H score analysis was performed using whole ovarian histologic sections. RESULTS: RT-PCR analysis showed the presence of mRNA for HCN1-4. Western blot analysis revealed HCN1-3 proteins in all ages of ovarian tissues. Immunohistochemistry with H score analysis demonstrated distinct age-related changes in patterns of HCN1-3 in the oocytes, granulosa cells, theca cells, and corpora lutea. HCN4 was present only in the oocytes, with declining levels during the reproduction lifespan. CONCLUSION: The evidence presented here demonstrates cell-type and developmental age patterns of HCN1-4 channel expression in rat ovaries. Based on this, we hypothesize that HCN channels have functional significance in rat ovaries and may have changing roles in reproductive aging.

Protection against cisplatin-induced ovarian damage by the antioxidant sodium 2-mercaptoethanesulfonate (mesna) in female rats.

OBJECTIVE: The hypothesis was that the administration of the antioxidant mesna (sodium 2-mercaptoethanesulfonate) during chemotherapy would protect ovaries against follicular damage. STUDY DESIGN: Sprague-Dawley rats were treated with saline solution, mesna-plus-cisplatin, or cisplatin. Immunohistochemistry was used to evaluate the Müllerian inhibiting substance (MIS) positive follicles. Serum and ovarian MIS were measured with enzyme-linked immunosorbent assay and Western blot analysis, respectively. Apoptosis in ovaries was studied by terminal deoxynucleotidyl transfer biotin-d UTP nick end labeling (TUNEL) assay. RESULT: Immunofluorescence staining for MIS was higher in preantral follicles in the mesna-plus-cisplatin group. The ovarian and serum MIS levels were higher in the mesna-plus-cisplatin than in the cisplatin alone group. There were no differences statistically in the TUNEL and the ovarian cyst analyses. CONCLUSION: Mesna, which was used at the time of cisplatin administration, protected ovaries against damage. The data that are presented challenge the existing clinical paradigm that gonadotropin-releasing hormone agonists represent the only medical method for the protection of ovaries during chemotherapy. Alternative medical means to protect ovaries during chemotherapy may be achievable.

Baseline and stimulated serum inhibin levels as biomarkers of cisplatin-induced ovarian damage in female rats.

OBJECTIVE: Chemotherapeutic agents, such as cisplatin, injure the ovary. It was hypothesized that serum inhibins can serve as biomarkers of ovarian damage from cisplatin. STUDY DESIGN: Adult female rats were administered cisplatin and afterward pregnant mare serum gonadotropin (PMSG) was given to stimulate ovarian inhibin production. RESULTS: At baseline, the serum inhibin A levels in the cisplatin-treated groups were slightly lower than saline. However, after PMSG stimulation, the cisplatin groups had dose-related and significantly blunted serum inhibin A and B responses. Results of Western blot and enzyme-linked immunosorbent assay inhibin analysis of ovarian lysate were consistent with the serum levels. Investigation of the ovaries showed that cisplatin groups at baseline had higher percentages of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling-positive follicles in inhibin positive follicles, suggesting that cisplatin induced apoptosis in the inhibin follicles. CONCLUSION: We conclude that serum inhibin A and B after PMSG ovarian challenge can be used as biomarkers to define rat ovarian damage after exposure to cisplatin.

Ovarian vascular endothelial growth factor and vascular endothelial growth factor receptor patterns in reproductive aging.

OBJECTIVE: To investigate how levels of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) change during corpus luteum formation in reproductive aging. DESIGN: Adult and reproductive aging female rats were given pregnant mare serum gonadotropin (PMSG) and the ovaries and sera were collected at 0, 3, 5, and 7 days after human chorionic gonadotropin (hCG) injection. SETTING: Laboratory study. PATIENT(S): Animal model. INTERVENTION(S): Induction of corpus luteum formation with PMSG-hCG. MAIN OUTCOME MEASURE(S): Western blot analysis for ovarian VEGF, VEGFR-1, VEGFR-2, and hypoxia-induced factor 1 (HIF1-alpha). Enzyme-linked immunosorbent assay (ELISA) measurement of serum VEGF levels. RESULT(S): For VEGF and VEGFR-1, there was no difference between the adult and the reproductive aging ovaries. For total VEGFR-2 levels, the adult ovaries had a decline during corpus luteum formation. In contrast, the levels of the two forms of phosphorylated VEGFR-2 declined in aging ovaries. There was a decline in HIF1-alpha during corpora lutea formation in the reproductive aging rats. CONCLUSION(S): The reproductive aging corpora lutea have a temporal decline in phosphorylated VEGFR-2 levels but not in the total VEGFR-2 levels. The data suggest that the VEGFR response mechanism is different in the aging ovary.

Serum and ovarian Müllerian inhibiting substance, and their decline in reproductive aging.

The objectives of this study were to identify whether there is a decline in Müllerian inhibiting substance (MIS) in the female rat during chronological aging, and to define the physiological basis of aging-related changes in MIS. The results demonstrate that there is an exponential decline in both serum and ovarian levels of MIS with increasing female age, and that the histologic origin for the reduction in serum levels of MIS is a decline in the number of small ovarian follicles expressing MIS.

Müllerian inhibiting substance as a novel biomarker of cisplatin-induced ovarian damage.

Müllerian inhibiting substance (MIS) has been investigated as a possible serum biomarker in human aging to estimate the number of female germ cells remaining. Cisplatin is an effective chemotherapeutic agent that is associated with ovarian injury. In this study, we tested the hypothesis that decreasing serum MIS can serve as a biomarker of ovarian damage after cisplatin. Adult female rats were treated with saline, 4.5, or 6.0 mg/kg cisplatin. The serum MIS levels were lower in both cisplatin groups, in a dose-related fashion. The ovarian lysates of both cisplatin groups had less MIS than control. Immunofluorescence analysis showed that the percentage of MIS-positive follicles was lower in the 6.0 mg/kg group. TUNEL assays showed that there was a dose related increase in the number of apoptotic follicles in the cisplatin groups. In summary, a decrease in serum MIS could serve as a biomarker to discriminate the degree of ovarian damage after cisplatin. These data are the first to establish in the rat that ovarian injury due to a chemotherapeutic agent could be monitored with the non-invasive serum biomarker MIS.