HOXA9 Overexpression Research Articles

METTL14-mediated HOXA5 m6A modification alleviates osteoporosis via promoting WNK1 transcription to suppress NLRP3-dependent macrophage pyroptosis

BackgroundOsteoporosis is a commonly diagnosed metabolic bone disease. NLRP3 inflammasome activation and pyroptosis are observed during osteoporosis. However, the mechanism by which NLRP3-mediated pyroptosis contributes to osteoporosis remains largely undefined. MethodsOvariectomized (OVX) mice were employed as an in vivo model of osteoclastogenesis. H&E staining and micro-CT detected the histological changes and bone parameters in the femur tissues. RANKL-treated macrophages were used as the in vitro model of osteoclastogenesis, and LPS/ATP treatment was used as the macrophage pyroptosis model. The cytotoxicity, cytokine secretion and caspase-1 activity were assessed by LDH release assay, ELISA and flow cytometry, respectively. The osteoclast formation ability was detected by TRAP staining. qRT-PCR, IHC and Western blotting detected the expression and localization of METTL14, pyroptosis-related or osteoclast-specific molecules in femur tissues or macrophages. Mechanistically, MeRIP assessed the m6A modification of HOXA5. Luciferase and ChIP assays were employed to detect the direct association between HOXA5 and WNK1 promoter in macrophages. ResultsMETTL14, HOXA5 and WNK1 were decreased in OVX mice, which was associated with pyroptosis. METTL14 or HOXA5 overexpression suppressed macrophage-osteoclast differentiation and pyroptosis, along with the upregulation of WNK1. METTL14-mediated m6A modification stabilized HOXA5 mRNA and increased its expression, and HOXA5 regulated WNK1 expression via direct binding to its promoter. Functional studies showed that WNK1 knockdown counteracted METTL14- or HOXA5-suppressed pyroptosis and macrophage-osteoclast differentiation. In OVX mice, overexpression of METTL14 or HOXA5 alleviated osteoporosis via suppressing WNK1-dependent NLRP3 signaling. ConclusionMETTL14-mediated HOXA5 m6A modification increased its expression, thereby inducing WNK1 expression and suppressing NLRP3-dependent pyroptosis to alleviate osteoporosis. The combination of METTL14 or HOXA5 agonist with pyroptosis targeted therapy may be a promising therapeutic approach for osteoporosis. The Translational Potential of this Article·•METTL14 or HOXA5 overexpression suppressed macrophage-osteoclast differentiation and pyroptosis in macrophages.·•METTL14-mediated m6A modification stabilized HOXA5 mRNA and increased its expression.•HOXA5 regulated WNK1 expression via direct binding to its promoter.•Silencing of WNK1 reversed METTL14- or HOXA5-suppressed pyroptosis and macrophageosteoclast differentiation.·•METTL14 or HOXA5 overexpression alleviated osteoporosis via suppressing WNK1-dependent NLRP3 signaling in OVX mice.

In Vivo Chemical Screening in Zebrafish Embryos Identified FDA-Approved Drugs That Induce Differentiation of Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is characterized by the abnormal proliferation and differentiation arrest of myeloid progenitor cells. The clinical treatment of AML remains challenging. Promoting AML cell differentiation is a valid strategy, but effective differentiation drugs are lacking for most types of AML. In this study, we generated Tg(drl:hoxa9) zebrafish, in which hoxa9 overexpression was driven in hematopoietic cells and myeloid differentiation arrest was exhibited. Using Tg(drl:hoxa9) embryos, we performed chemical screening and identified four FDA-approved drugs, ethacrynic acid, khellin, oxcarbazepine, and alendronate, that efficiently restored myeloid differentiation. The four drugs also induced AML cell differentiation, with ethacrynic acid being the most effective. By an RNA-seq analysis, we found that during differentiation, ethacrynic acid activated the IL-17 and MAPK signaling pathways, which are known to promote granulopoiesis. Furthermore, we found that ethacrynic acid enhanced all-trans retinoic acid (ATRA)-induced differentiation, and both types of signaling converged on the IL-17/MAPK pathways. Inhibiting the IL-17/MAPK pathways impaired ethacrynic acid and ATRA-induced differentiation. In addition, we showed that ethacrynic acid is less toxic to embryogenesis and less disruptive to normal hematopoiesis than ATRA. Thus, the combination of ethacrynic acid and ATRA may have broader clinical applications. In conclusion, through zebrafish-aided screening, our study identified four drugs that can be repurposed to induce AML differentiation, thus providing new agents for AML therapy.

HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes

BackgroundHox gene family is an important transcription factor that regulates cell process, and plays a role in the process of adipocytes differentiation and fat deposition. Previous transcriptome sequencing studies have indicated that the Homeobox A9 gene (HOXA9) is a candidate gene for regulating the process of bovine lipid metabolism, but the function and specific mechanism of action remain unclear. Therefore, this study aims to explore the role of HOXA9 in the proliferation, differentiation and apoptosis of bovine preadipocytes through gain-of-function and lose-of-function.ResultIt found HOXA9 highly expressed in bovine adipose tissue, and its expression level changed significantly during adipocytes differentiation process. It gave a hint that HOXA9 may be involved in the process of bovine lipid metabolism. The results of HOXA9 gain-of-function experiments indicated that HOXA9 appeared to act as a negative regulator not only in the differentiation but also in the proliferation of bovine preadipocytes, which is mainly reflected that overexpression of HOXA9 down-regulate the mRNA and protein expression level of PPARγ, CEBPα and FABP4 (P < 0.05). The mRNA expression level of CDK1, CDK2, PCNA, CCNA2, CCNB1, CCND1 and CCNE2, as well as the protein expression of CDK2 also significantly decreased. The decrease of lipid droplets content was the main characteristic of the phenotype (P < 0.01), which further supported the evidence that HOXA9 was a negative regulator of preadipocytes differentiation. The decrease of cell proliferation rate and EdU positive rate, as well as the limitation of transition of preadipocytes from G0/G1 phase to S phase also provided evidence for the inhibition of proliferation. Apart from this above, we noted an interesting phenomenon that overexpression of HOXA9 showed in a significant upregulation of both mRNA and protein level of apoptosis markers, accompanied by a significant increase in cell apoptosis rate. These data led us not to refute the fact that HOXA9 played an active regulatory role in apoptosis. HOXA9 loss-of-function experiments, however, yielded the opposite results. Considering that HOXA9 acts as a transcription factor, we predicted its target genes. Dual luciferase reporter assay system indicated that overexpression of HOXA9 inhibits activity of PCNA promoter.ConclusionTaken together, we demonstrated for the first time that HOXA9 played a role as a negative regulatory factor in the differentiation and proliferation of preadipocytes, but played a positive regulatory role in apoptosis, and it may play a regulatory role by targeting PCNA. This study provides basic data for further exploring the regulatory network of intramuscular fat deposition in bovine.

A novel t(X;21)(p11.4;q22.12) translocation adds to the role of BCOR and RUNX1 in myelodysplastic syndromes and acute myeloid leukemias.

In myeloid neoplasms, both fusion genes and gene mutations are well-established events identifying clinicopathological entities. In this study, we present a thus far undescribed t(X;21)(p11.4;q22.12) in five cases with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The translocation was isolated or accompanied by additional changes. It did not generate any fusion gene or gene deregulation by aberrant juxtaposition with regulatory sequences. Molecular analysis by targeted next-generation sequencing showed that the translocation was accompanied by at least one somatic mutation in TET2, EZH2, RUNX1, ASXL1, SRSF2, ZRSR2, DNMT3A, and NRAS genes. Co-occurrence of deletion of RUNX1 in 21q22 and of BCOR in Xp11 was associated with t(X;21). BCOR haploinsufficiency corresponded to a significant hypo-expression in t(X;21) cases, compared to normal controls and to normal karyotype AML. By contrast, RUNX1 expression was not altered, suggesting a compensatory effect by the remaining allele. Whole transcriptome analysis showed that overexpression of HOXA9 differentiated t(X;21) from both controls and t(8;21)-positive AML. In conclusion, we characterized a new recurrent reciprocal t(X;21)(p11.4;q22.12) chromosome translocation in MDS and AML, generating simultaneous BCOR and RUNX1 deletions rather than a fusion gene at the genomic level.

HOXA10 promotes Gdf5 expression in articular chondrocytes

Growth differentiation factor 5 (GDF5), a BMP family member, is highly expressed in the surface layer of articular cartilage. The GDF5 gene is a key risk locus for osteoarthritis and Gdf5-deficient mice show abnormal joint development, indicating that GDF5 is essential in joint development and homeostasis. In this study, we aimed to identify transcription factors involved in Gdf5 expression by performing two-step screening. We first performed microarray analyses to find transcription factors specifically and highly expressed in the superficial zone (SFZ) cells of articular cartilage, and isolated 11 transcription factors highly expressed in SFZ cells but not in costal chondrocytes. To further proceed with the identification, we generated Gdf5-HiBiT knock-in (Gdf5-HiBiT KI) mice, by which we can easily and reproducibly monitor Gdf5 expression, using CRISPR/Cas9 genome editing. Among the 11 transcription factors, Hoxa10 clearly upregulated HiBiT activity in the SFZ cells isolated from Gdf5-HiBiT KI mice. Hoxa10 overexpression increased Gdf5 expression while Hoxa10 knockdown decreased it in the SFZ cells. Moreover, ChIP and promoter assays proved the direct regulation of Gdf5 expression by HOXA10. Thus, our results indicate the important role played by HOXA10 in Gdf5 regulation and the usefulness of Gdf5-HiBiT KI mice for monitoring Gdf5 expression.

LSD1 Inhibition Enhances Antigen Presentation and Co-Stimulation in AML to Promote T Cell-Mediated Anti-Leukemia Immune Responses

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide a curative treatment option for acute myeloid leukemia (AML). Donor T cells play an important role in the curative potential of allo-HSCT by recognizing and eliminating residual leukemic cells, inducing the desirable graft-versus-leukemia (GVL) effect. Evading from this effect of donor T cell immune surveillance through the downregulation of human leukocyte antigen class II (HLA-II) molecules has been shown to contribute to AML relapse after allo-HSCT. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML due to its role in regulating myeloid differentiation pathways. We hypothesized that LSD1 inhibition has the potential to enhance anti-leukemic immune responses by differentiating AML cells and inducing the acquisition of markers typical for antigen-presenting cells. We have studied these effects by treating human AML cell lines with the LSD1 inhibitor bomedemstat and investigated the expression of antigen-presenting molecules by flow cytometry and quantitative PCR (qPCR). At a concentration of 100 nM, bomedemstat upregulated the expression of CIITA by a log2 fold change of 2.23 ± 0.68 (p &amp;lt; 0.01) in OCI-AML3, 2.17 ± 0.28 (p &amp;lt; 0.01) in HL60 and 1.80 ± 0.67 (p &amp;lt; 0.05) in MOLM-13. To assess if the observed upregulation of CIITA can lead to a re-expression of HLA-DR in these HLA-DR negative cell lines, we then treated these cell lines with 100 nM of bomedemstat with additional IFN-γ stimulation. Expression of HLA-DR and the co-stimulatory molecule CD86 was determined by flow cytometry. Bomedemstat increased the expression of HLA-DR and CD86 across all three cell lines leading to the generation of 26.63 ± 3.07% (p &amp;lt; 0.0001) HLA-DR +/CD86 + cells in OCI-AML3, 4.19 ± 1.68% (p &amp;lt; 0.001) HLA-DR +/CD86 + cells in HL-60 and 14.67 ± 2.03% (p &amp;lt; 0.001) HLA-DR +/CD86 + cells in MOLM-13. We further assessed the effects of bomedemstat treatment on cytokine production by qPCR. Notably, bomedemstat treatment increased the expression of CXCL-10 by a log2 fold change of 2.59 ± 0.66 (p &amp;lt; 0.001) in MOLM-13 and 4.12 ± 0.88 (p &amp;lt; 0.001) in HL-60 at 100 nM concentration. The upregulation of CXCL-10 was also validated using bead-based ELISA assays. To investigate the molecular mechanism of the observed concomitant upregulation of MHC-II molecules and CD86, we then used murine myeloid progenitor cells transformed by overexpression of Hoxa9 and Meis1 (H9M) or MN1. In H9M-transformed cells, bomedemstat concurrently upregulated MHC-II (I-A/I-E) and CD86 even in the absence of IFN-γ stimulation, leading to an increase in MHC-II +/CD86 + cells from 2.52 ± 1.22% at baseline to 29.95 ± 14.11 (p &amp;lt; 0.05). This effect was not observed in MN1-transformed cells. H9M-transformed progenitor cells from Irf8 knockout mice (H9M- Irf8-KO) did not show any upregulation of MHC-II, pointing towards a role for Irf8 in this process. To interrogate whether the concurrent upregulation of MHC-II and CD86 can enhance CD4 + T cell activation, we used ovalbumin-expressing H9M-transformed cells that were co-cultured with OT-II T cells after bomedemstat treatment. We could show that bomedemstat-treatment enhanced the ability of H9M-OVA cells to activate T cells as demonstrated by an enhanced expression of the T cell activation markers CD25 and CD69. Furthermore, bomedemstat also increased the immune killing of OVA-peptide pulsed H9M cells at an effector-to-target (E:T) ratio of 1:1 from 1.12 ± 4.01 to 19.70 ± 6.20% (p &amp;lt; 0.01). It also enhanced the killing of OVA-expressing H9M cells from 5.80 ± 2.95% to 17.70 ± 3.16% (p &amp;lt; 0.05). No significant killing effect was observed for MN1 cells. In conclusion, we demonstrate both phenotypically and functionally that LSD1 inhibition by bomedemstat can enhance the immunogenicity of human and murine AML models. These findings point towards a potential role of LSD1 inhibition as a maintenance therapy after allo-HSCT.

Ubtf tandem Duplications Define a Novel Subtype of Acute Myeloid Leukemia Associated with Younger Age, WT1 Mutations and HOXA9 Marked Overexpression

Background Upstream Binding Transcription Factor ( UBTF) gene plays an important role in ribosomal RNA transcription. UBTF tandem duplications (TD) have been recently described as a recurrent genetic lesion in acute myeloid leukemia (AML). UBTF-TDs appear to be mutually exclusive with other class-defining lesions in AML and display a unique comutational signature. UBTF-mutated AML affects predominantly younger patients and is associated with trisomy 8, internal tandem duplication of FLT3 ( FLT3-ITD), mutations in WT1 and poor prognosis. Moreover, HOX and MEIS genes seem to be overexpressed in AML with UBTF-TD similarly to NPM1-mutated ( NPM1m) or KMT2A-rearragend ( KMT2Ar) AML, suggesting potential sensitivity to menin inhibitors. In this study we sought to characterize UBTF-TDs in our AML cohort. Methods We studied 456 adult patients (median age 54 years; range 18-70) diagnosed with de novo AML and included in AML-12 protocol from CETLAM group. All patients from the 2012-2017 period (n=403) were consecutively studied and we also selected 53 cases of WT1-mutated AML from the 2017-2020 period, given the strong association with UBTF-TD. Screening for UBTF-TD was performed on bone marrow (BM) or peripheral blood (PB) genomic DNA samples. Exon 13 of UBTF gene was amplified and analysed by Polymerase chain reaction (PCR) and subsequent fragment length analysis by capillary electrophoresis. Mutated samples were confirmed through Sanger sequencing. Further, RNA from 9 UBTF-TD cases was available and HOXA9 and MEIS1 gene expression analysis was performed by Real-Time Polymerase Chain Reaction (RT-qPCR). Clinical and biological data were obtained from CETLAM registry. Results UBTF-TD was found in 13 patients from our cohort whose characteristics are detailed in Table 1. UBTF-TDs range from 45 base pairs (bp) to 365 bp, being the 48 bp the most recurring size. As described, although small UBTF-TDs are in frame insertions, the largest ones are not multiple of 3. Notably, most UBTF-TDs presented small insertions and deletions among the duplication. Presence of UBTF-TD was associated with younger age (36 vs 55 years; p&amp;lt;0.001) and BM dysplastic changes (80% of U BTFMUT). UBTF-TD was particularly frequent (8.9% of all cases) in patients below 40 years, compared to other lesions as CEBPA mutations (1.1%). Eleven (85%) U BTFMUT patients presented intermediate cytogenetics, being trisomy 8 (5), normal karyotype (3) and 9q deletion (2) the most recurrent findings. Two cases of adverse karyotype with MECOM-rearrangements were found. As previously described, there was a strong association between UBTF-TD and of FLT3-ITD (38.5%). Other recurrent genetic lesions in AML like NPM1 and CEBPA mutations or core-binding factor (CBF) rearrangements were exclusive of UBTF wild type patients. Further, we found that UBTFMUT leukemias showed marked overexpression of HOXA9 compared with other AML subtypes (22.1-fold vs 5.0-fold; p&amp;lt;0.001; Fig. 1) and similar to NPM1m AML (22.3-fold). On the other hand, MEIS expression did not differ significantly among groups. Conclusions In our cohort of adult AML patients, UBTF-TDs are found in 1.8% of cases and are especially frequent in younger individuals in absence of other class-defining genetic lesions. The presence of UBTF-TD is strongly associated with cytomorphological dysplasia and co-occurrence of FLT3-ITD and WT1 mutations, while NPM1 and CEBPA mutations and CBF rearrangements appear to be mutually exclusive. HOXA9 upregulation could be a leukemogenic event in UBTF-TD AML, as described in NPM1-m and KMT2A-r AML, suggesting potential utility of menin inhibitors in this subset of patients. Screening for UBTF-TD is going to be incorporated in our molecular diagnostic panel for new AML cases.

RNA-Binding Protein RBM5 Plays an Essential Role in Acute Myeloid Leukemia By Activating the Oncogenic Protein HOXA9

Acute myeloid leukemia (AML) represents a type of malignant hematological disease that indicates poor outcomes in children and adults. HOXA9 overexpression is observed in 50-70% of human AML and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include but are not limited to those carrying KMT2A gene rearrangements (KMT2A-r), NPM1c mutations, NUP98-fusions and EZH2 mutation. Accumulating genetic evidence also indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation, and loss of function of HOXA9 and downstream pathways consequently impaired leukemia maintenance. Although HOXA9 is not an ideal direct candidate for therapeutic interventions, uncovering the novel regulators of HOXA9 might elucidate the HOXA9 regulation mechanism that could be exploited to identify novel therapeutic targets in HOXA9-driven leukemias. In this study, we performed genome-wide CRISPR/Cas9 screens in the HOXA9-endogenous reporter acute leukemia cells and unbiasedly identified that candidate genes are significantly enriched in RNA binding and RNA splicing regulation. An RBP protein, RBM5, was among the top essential genes. Initially, RBM5 was reported as a tumor suppressor gene in lung cancer. Thus far, the function of the encoding protein RBM5 in blood cancer remained largely unknown. To further test how RBM5 expression might correlate with AML development, we analyzed the public RNA-seq data from the TCGA clinical cancer sample datasets, RBM5 is highly expressed in AML, indicating a plausible role in AML oncogenesis. Functional validation by two individual sgRNAs against different exons of RBM5 confirmed the selectively survival essential of RBM5 in AML cell lines. Ectopic expression of sgRNA-resistant RBM5 cDNA ultimately rescued the cellular phenotype upon RBM5 loss. We utilized an alternative loss-of-function approach, RNA interference (RNAi), to further examine the functional requirement of RBM5, Two independent shRNAs targeted RBM5 mRNA in three AML cells lines. RBM5 loss significantly impaired cell growth, reduced colony numbers and triggered notable myeloid differentiation. We further transplant MOLM13 cells targeted with shRBM5 into NSG mice, and observed that RBM5 shRNAs-treated animals lagged progressively more behind with much less splenomegaly and reduced leukemia cell infiltration in bone marrow, spleen, and peripheral blood compared to the wild-type control, suggesting that RBM5 is required for AML progression in vivo. More importantly, Suppression of RBM5 in primary AML cells significantly reduced colony numbers and induced myeloid differentiation, as demonstrated by the maturation markers CD11b and CD14. In addition, the domain dropout CRISPR screen revealed that the DNA-binding domain C2H2 is required for the gene function. Integrative analysis further suggested that HOXA9 promises the direct transcriptional target of RBM5 as demonstrated by functional rescue assay and HOXA9 partially restore the gene transcriptional defect in RBM5 null leukemia cells. In summary, our innovative research reported that a new RNA-binding protein, RBM5, is required for the survival of AML through non-canonical transcriptional regulation via a complex regulatory mechanism. These data support RBM5 as a promising AML therapeutic target in future studies.

Is HOXA5 a Novel Prognostic Biomarker for Uterine Corpus Endometrioid Adenocarcinoma?

Endometrial cancer (EC) is one of the most pervasive malignancies in females worldwide. HOXA5 is a member of the homeobox (HOX) family and encodes the HOXA5 protein. HOXA5 is associated with various cancers; however, its association with EC remains unclear. This study aimed to determine the association between HOXA5 gene expression and the prognosis of endometrioid adenocarcinoma, a subtype of EC (EAEC). Microarray data of HOXA5 were collected from the Gene Expression Omnibus datasets, consisting of 79 samples from GSE17025 and 20 samples from GSE29981. RNA-sequencing, clinical, and survival data on EC were obtained from The Cancer Genome Atlas cohort. Survival analysis revealed that HOXA5 overexpression was associated with poor overall survival in patients with EAEC (p = 0.044, HR = 1.832, 95% CI = 1.006-3.334). Cox regression analysis revealed that HOXA5 was an independent risk factor for poor prognosis in EAEC. The overexpression of HOXA5 was associated with a higher histological grade of EAEC, and it was also associated with TP53 mutation or the high copy number of EC. Our findings suggest the potential of HOXA5 as a novel biomarker for predicting poor survival outcomes in patients with EAEC.

Abstract 6077: Comprehensive multi-omic characterization of genetic changes associated with sinonasal squamous cell carcinoma progression

Abstract Approximately 50-80% of sinonasal tract cancers are sinonasal squamous cell carcinoma (SNSCC), a rare malignancy which occur in the nasal cavity and maxillary sinus. These lesions possess a propensity for local invasion into adjacent structures including the skull base, brain, and orbit. Owing to relatively mild symptoms during early stages, which resemble benign sinonasal disease, most patients are already in clinically advanced stages when diagnosed. Therefore, despite improvements in endoscopic surgical approaches, radiotherapy and imaging techniques, the 5-year overall survival for advanced SNSCC patients remains poor (~40%). Inverted sinonasal papilloma (IP) is a locally aggressive, benign epithelial neoplasm arising in the paranasal sinuses which has a high recurrence rate and transforms to SNSCC in 10%-25% of cases. As some IP evolve to malignant neoplasms, it has been presumed that they represent an intermediate step in SNSCC progression. However, the current tools for detecting altered epithelial cells, such as clinical examination and histological characteristics, have limited prognostic value for predicting which IP will progress to malignancy, and the link between IP and invasive disease remains unclear. Activating mutations in EGFR and PIK3CA genes as well as loss-of-function mutations in TP53 have been reported in a few very small cohorts of IP and SNSCC lesions, suggesting their role in IP pathogenesis and progression to invasive malignancy. Other observed alterations potentially associated with malignant transition include KRAS mutations, loss of heterozygosity (LOH) at the 9p21 locus (containing p16INK4a), mismatch repair genes deficiency, amplification of FGFR1 and SOX2 genes, as well as NFκB, COX2 and HOXA9 overexpression. While these studies provide a snapshot of molecular changes in SNSCC, genetic alterations critical to the development of SNSCC are poorly understood, and the exact mechanism underlying malignant transformation remains unknown. In this study we have used PandaOmic, a multi-omic data analysis and visualization algorithm, to perform a first comprehensive integrative analysis of WES, mitochondrial sequencing and transcriptomic data obtained from 11 IP lesions, matched invasive SNSCC tumors and histologically normal paranasal sinus epithelium collected from the same patients. Our analysis reveals heterogeneous mutational patterns, gene expression changes and signaling pathways features associated with SNSCC pathogenesis, and provides crucial insights that may aid in the development of novel means of prevention, diagnosis, and treatment of this rare, aggressive and poorly characterized malignancy. Citation Format: Alka Singh, Nikita Babushkin, Michael Korzinkin, Viktoria Sarkisova, Vasudha Mishra, Ari Rosenberg, Mark Lingen, Justin Bishop, Alex Zhavoronkov, Nyall London, Nishant Agrawal, Xuanyao Liu, Evgeny Izumchenko. Comprehensive multi-omic characterization of genetic changes associated with sinonasal squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6077.

Impact of T-cell Receptor Status on Mutational Landscape and Outcome in T-ALL.

Impact of T-cell Receptor Status on Mutational Landscape and Outcome in T-ALL.

Cell cycle, apoptosis, cell differentiation, and lipid metabolism gene expression in endometriotic tissue and exposure to parabens and benzophenones

AimTo describe the expression profile in endometriotic tissue of genes involved in four signaling pathways related to the development and progression of endometriosis (cell cycle, apoptosis, cell differentiation and lipid metabolism) and to explore its relationship with the women exposure to chemicals with hormonal activity released from cosmetics and personal care products (PCPs). MethodsThis cross-sectional study, encompassed within the EndEA study, comprised a subsample of 33 women with endometriosis. Expression levels of 13 genes (BMI1, CCNB1, CDK1, BAX, BCL2L1, FOXO3, SPP1, HOXA10, PDGFRA, SOX2, APOE, PLCG1 and PLCG2) in endometriotic tissue and urinary concentrations of 4 paraben (PB) and 3 benzophenone (BP) congeners were quantified. Bivariate linear and logistic regression analyses were performed to explore the associations between exposure and gene expression levels. ResultsA total of 8 out 13 genes (61.5 %) were expressed in >75 % of the samples. Exposure to congeners of PBs and/or BPs was associated with the overexpression of CDK1 gene (whose protein drives cells through G2 phase and mitosis), HOXA10 and PDGFRA genes (whose proteins favor pluripotent cell differentiation to endometrial cells), and APOE (whose protein regulates the transport and metabolism of cholesterol, triglycerides and phospholipids in multiple tissues) and PLCG2 genes (whose protein creates 1D-myo-inositol 1,4,5-trisphosphate and diacylglycerol, two important second messengers). ConclusionsOur findings suggest that women exposure to cosmetic and PCP-released chemicals might be associated with the promotion of cell cycle and cell differentiation as well as with lipid metabolism disruption in endometriotic tissue, three crucial signaling pathways in the development and progression of endometriosis. However, further studies should be accomplished to confirm these preliminary data.

Hox genes are crucial regulators of periosteal stem cell identity.

Periosteal stem and progenitor cells (PSPCs) are major contributors to bone maintenance and repair. Deciphering the molecular mechanisms that regulate their function is crucial for the successful generation and application of future therapeutics. Here, we pinpoint Hox transcription factors as necessary and sufficient for periosteal stem cell function. Hox genes are transcriptionally enriched in periosteal stem cells and their overexpression in more committed progenitors drives reprogramming to a naïve, self-renewing stem cell-like state. Crucially, individual Hox family members are expressed in a location-specific manner and their stem cell-promoting activity is only observed when the Hox gene is matched to the anatomical origin of the PSPC, demonstrating a role for the embryonic Hox code in adult stem cells. Finally, we demonstrate that Hoxa10 overexpression partially restores the age-related decline in fracture repair. Together, our data highlight the importance of Hox genes as key regulators of PSPC identity in skeletal homeostasis and repair.

HOXA5 inhibits the proliferation and metastasis of cervical squamous cell carcinoma by suppressing the β-catenin/Snail signaling.

HOXA5, as a transcription factor, plays an important role in a variety of malignant tumors. Nevertheless, its biological role in cervical squamous cell carcinoma (CSCC) is largely unknown. In our study, we aimed to explore the function of HOXA5 in CSCC and its molecular mechanism. Immunohistochemistry showed that HOXA5 expression was downregulated in human CSCC tissues and HOXA5 staining was negatively correlated with tumor size and histological grade of CSCC. Ectopic expression of HOXA5 inhibited proliferative and metastatic abilities of CSCC cells in vitro and in vivo. Furthermore, overexpression of HOXA5 inhibited the cell cycle by arresting the S/G2 phase by flow cytometry and that was related to the downregulation of Cyclin A. Further study showed that HOXA5 suppressed EMT by inhibiting the β-catenin/Snail signaling resulting in reduced metastasis of CSCC cells. Altogether, our results suggested that HOXA5 inhibited the proliferation and metastasis via repression of the β-catenin/Snail pathway, proposing the potential role of HOXA5 in the prevention and treatment of CSCC.

Knockdown of RFC4 inhibits the cell proliferation of nasopharyngeal carcinoma in vitro and in vivo.

Nasopharyngeal carcinoma (NPC) is a malignant tumor that mainly occurs in East and Southeast Asia. Although patients benefit from the main NPC treatments (e.g., radiotherapy and concurrent chemotherapy), persistent and recurrent diseases still occur in some NPC patients. Therefore, investigating the pathogenesis of NPC is of great clinical significance. In the present study, replication factor c subunit 4 (RFC4) is a key potential target involved in NPC progression via bioinformatics analysis. Furthermore, the expression and mechanism of RFC4 in NPC were investigated in vitro and in vivo. Our results revealed that RFC4 was more elevated in NPC tumor tissues than in normal tissues. RFC4 knockdown induced G2/M cell cycle arrest and inhibited NPC cell proliferation in vitro and in vivo. Interestingly, HOXA10 was confirmed as a downstream target of RFC4, and the overexpression of HOXA10 attenuated the silencing of RFC4-induced cell proliferation, colony formation inhibition, and cell cycle arrest. For the first time, this study reveals that RFC4 is required for NPC cell proliferation and may play a pivotal role in NPC tumorigenesis.

Loss of Cell Fate Determinant LLGL1 Impairs Leukemia Development in Acute Myeloid Leukemia

Cell fate determinants and polarity regulators influence the fine balance between self-renewal and differentiation in HSC and leukemic stem cells (LSC). Previously our group provided first evidence that inactivation of the cell fate regulator Llgl1 in murine HSCs leads to alterations in self-renewal, proliferative capacity and differentiation (Heidel et al., 2013). The impact of Llgl1 on malignant transformation and especially acute myeloid leukemia (AML) development had not been investigated in detail. Inactivation of LLGL1 in several human AML cell lines (MOLM13, HEL, MV4;11, HL-60) by CRISPR/Cas9 resulted in decreased proliferation of leukemic cells. Consistently, xenograft studies using LLGL1-depleted human AML cells resulted in delayed disease development compared to non-targeting control (median survival NT-control: 37.5 days vs. LLGL1-KD: 92 days; p=0.0002). Genetic inactivation of LLGL1 in primary AML patient cells resulted in reduced colony formation in vitro and improved survival of recipient mice in a patient-derived xenograft model of AML. To recapitulate these findings in defined model systems of murine AML, we used a conditional Llgl1 knockout mouse model (Klezovitch et al, 2004) and intercrossed it with AML-models harboring different driver mutations: MllAF9 (Cerral et al., 1995) or Flt3ITD/ITD (Lee et al., 2006). Conditional deletion of Llgl1 in MllAF9 knockin mice using an Mx1-Cre-recombinase (Cre+) resulted in significant delay of leukemia development (median survival +/+: 174 days; -/-: 203 days; p=0.0361). To confirm these findings, we investigated a second oncogenic model and induced deletion of Llgl1 in Flt3ITD/ITD knockin mice. Leukemic Flt3ITD/ITD; Llgl1+/+; Cre+ animals (n=12) showed rapid onset of leukemia development, whereas the Flt3ITD/ITD; Llgl1-/-; Cre+ animals (n=25) showed significant delay in leukemia progression (median survival +/+: 38 days; -/-: 51 days; p=0.0005). Llgl1-/- animals show reduced peripheral blast counts (WBC +/+: 332.558 Gpt/l; -/-: 141.083 Gpt/l; p=0.0012) and decreased organ infiltration. Importantly, immunophenotypic analysis of bone marrow (BM) compartments indicated a shift of immature cells towards a more differentiated state. The overall abundance of leukemic Lin-Kit+Sca-1+ (LSK) cells was reduced in Flt3ITD/ITD; Llgl1-/- animals compared to Flt3ITD/ITD; Llgl1+/+ controls (LSK +/+: 13099±3144/106 BMC; -/-: 8117±2810 /106 BMC; p=0.0227). In contrast, we found increased numbers of Lin-Kit+Sca-1+FcgR+CD34+ of leukemic granulocyte-macrophage progenitors (LGMPs: +/+: 8306±1963/106 BMC; -/-: 14755±4783/106 BMC; p=0.0141) upon deletion of Llgl1 in Flt3ITD/ITD animals. These findings indicate that genetic inactivation of Llgl1 results in loss of leukemia stemness and shifts the leukemia stem cell phenotype from HSC-like to GMP-like LSCs. To test for this hypothesis, we performed global transcriptome analysis of sorted LSK cells of Flt3ITD/ITD mice early after genetic deletion of Llgl1. Among the significantly downregulated genes, we found several members of the HoxA-cluster, consistent with the immunophenotypic loss of stemness. Gene-set-enrichment analysis revealed downregulation of leukemia stem cell signatures (Gal et al., 2006) and enrichment of a gene set associated with LGMPs (compared to leukemic-HSC) signatures (Krivtsov et al., 2006). To validate whether loss of Hox-gene expression is relevant for impaired leukemia development, we performed rescue experiments in vivo. LSK cells derived from Flt3ITD/ITD; Llgl1+/+ or Flt3ITD/ITD; Llgl1-/- animals were retrovirally transduced with HoxA9 or empty vector control (eV). When transplanted into sublethally irradiated recipient mice, overexpression of HoxA9 (OE) in Llgl1-/-; Flt3ITD/ITD cells rescued the disease phenotype comparable to Flt3ITD/ITD; Llgl1+/+ cells (median survival +/+;eV: 62.50%; -/-; OE: 55.56%). In contrast, empty vector control Flt3ITD/ITD; Llgl1-/- recipients showed no disease development (median survival -/-; eV: 100%). Taken together our results may provide first evidence for a functional role of Llgl1 in models of AML. In contrast to its function in normal HSCs, deletion of Llgl1 appears to reduce stemness of leukemic stem cells and impair leukemia development in vitro and in vivo. Experiments to assess for the effects of Llgl1 on LSC polarity and cell fate decisions are currently under way.

Systematically Interrogating Downstream Regulation Mechanisms of HOXA9 in MLL-r Leukemia Via Functional Noncoding CRISPR Screens

HOXA9 overexpression is observed in 50-70% of human acute myeloid leukemia (AML) and a subset of acute lymphoblastic leukemia (ALL) and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include those carrying MLL gene rearrangements (MLL-r), NPM1c mutations, and other genetic alterations. Frequent genetic translation of NUP98-HOXA9 retained the DNA binding domain of HOXA9, suggesting the transcription factor function is essential for leukemia initiation and maintenance. Accumulating evidence indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation. However, HOXA9 protein itself is a poor therapeutic target as it lacks targetable binding domains. Therefore, understanding HOXA9's functional downstream targets will provide alternative therapeutic targets. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes in MLL-r leukemia and across leukemias of other genetic subtypes. In this study, we have successfully established a HOXA9-miniAID expressing MLL-r B-ALL cell line to execute acute HOXA9 degradation in the same isogenic cell line background upon auxin treatment. Compared with conventional loss-of-function strategies, our AID cellular model allows the investigation of immediate downstream targets controlled by HOXA9. We have therefore identified about 1,800 reproducible peaks bound by HOXA9 at a genome-wide scale. About 80% of HOXA9 binding peaks were co-bound by HOXA9's binding partner MEIS1, and motif enrichment confirmed that more than 75% of peaks contained a typical HOXA9 consensus motif. Interestingly, >80% binding peaks are located in non-promoter cis-regulator elements. Given the essential role of HOXA9 in regulating MLL-r leukemia cell survival and differentiation, we reasoned that targeting specific HOXA9-bound peaks will affect downstream gene expression leading to a similar survival crisis seen in HOXA9 knockout cells. Based on this rationale, combinatorial CRISPR screens were performed in three biological replicates on the MLL-r SEM cell line stably expressing Cas9 and base editor (ABE8.0), followed by time-course dropout selection on days 7, 14, and 21. Integrative CRISPR screen analysis identified six reproducible noncoding hits, including a positive control located in the distal enhancer of FLT3. Functional validation by targeting these six noncoding segments with Cas9, dCas9-KRAB in additional MLL-r leukemia cell lines MOLM13, MV4,11 confirmed the survival essential of these HOXA9-bound regions. No cellular phenotype was observed in non-MLL-r leukemia cell lines, including NALM6, JURKAT, or OCI-AML2. Transplanting SEM cells targeted with sgHOXA9-in-FLT3 into NSG mice, significant growth retardation was monitored by flow analysis of peripheral blood. In addition, RNA-seq and Q-PCR analysis further identified the functional relevant downstream genes upon CRISPR editing of these HOXA9-bound noncoding segments, including FLT3, XBP1, JUN, BAHCC1, and CCDC200. Chromatin conformation characterization by Capture-C further confirmed the long-range interaction between HOXA9 bound enhancers and corresponding gene promoters in MLL-r SEM cells. In summary, using state-of-art research tools and newly established cell models, we have conducted a systematic functional screen and mechanism study of an undocumented downstream transcriptional network of HOXA9 in MLL-r leukemia cells. The work is significant as it will advance our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency. Moreover, our study will promote the future development of alternative therapeutic targets in HOXA9-driven (including HOXA9 and possibly NUP98-HOXA9 subtype) leukemia in patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting SAMHD1 Promotes Anti-Tumor Immunity in Acute Myeloid Leukemia

The overall outcome of AML, which is characterized by HOXA9 overexpression (>50% cases), remains unfavorable. Success of allogeneic hematopoietic stem cell transplantation and immunotherapy highlights that T cell activation is critical for AML eradication. Notably, suppression of T cell immunity is common in the AML BM microenvironment, thus there is a pressing need to define underlying mechanisms. Recent studies reveal that the dNTP and Ara-CTP (active form of cytarabine) hydrolase SAMHD1, whose loss-of-function mutation is seen in inflammatory encephalopathy named Aicardi-Goutières syndrome, limits type-1 Interferon (IFN-I) and T cell responses by suppressing cGAS/STING signaling. Interestingly, our analysis of GEO datasets showed elevated SAMHD1 levels in specimens from AML patients relative to healthy donors, correlated with HOXA9 expression (A) and decreased T cell function. We thus asked whether SAMHD1 levels are associated with a particular cytogenetic change or mutation upstream of HOXA9 dysregulation. Accordingly, analysis of GSE data demonstrated that AML cases with MLL rearrangement (MLL-r, t[11q23]) or NPM1 mutation (NPM1c) exhibited elevated SAMHD1 levels relative to those without either alteration (B, C). Notably, HOXA9 knockdown (KD) decreased SAMHD1 protein levels in Molm13 (MLL-r+) and OCI-AML3 (NPM1c+) cells. ChIP-Q-PCR analysis of cord blood CD34+ cells engineered with Flag-tagged HoxA9 showed significant enrichment of HoxA9 protein at the predicted SAMHD1 promoter region relative to a control site. That region also showed significant enrichment of H3K27Ac (an active transcription mark) in HoxA9-overexpressing relative to control cells. We next assessed SAMHD1 loss-of-function in MLL-AF9 (MA9) knock-in mice, first by transducing leukemia cells from MA9+ BM with a doxycycline (DOX)-inducible control or shSAMHD1 and then transplanting them into either WT immunocompetent or immunodeficient (Rag2-/-) recipients to induce leukemia. Following DOX treatment, Rag2-/- mice bearing SAMHD1-KD or control transplants exhibited comparable survival. However, WT recipients bearing SAMHD1-KD AML survived significantly longer than controls (D, E). Moreover, SAMHD1-KD-mediated leukemia ablation was reproduced in HOXA9-Meis1 transduction/transplantation AML. Furthermore, depletion of T cells, but not B cells, blocked survival advantages seen in SAMHD1-KD-transplanted animals. To determine if IFN-I responses underlie these effects, we implanted SAMHD1-KD MA9 cells into WT or Ifnar1-/- recipients and observed significant abolition of SAMHD1-KD anti-AML effects. Moreover, SAMHD1-KD induced cGAS activation, as evidenced by elevated levels of the immune transmitter cGAMP in BM fluid from SAMHD1-KD relative to control MA9 mice. Accordingly, we observed upregulation of multiple interferon-stimulated genes (ISGs) such as ISG15 and CXCL10 downstream of cGAS/STING after SAMHD1-KD, effects blocked by cGAS deletion. To determine if immune activation requires SAMHD1 catalysis, we knocked out endogenous SAMHD1 in THP1 cells, which we then engineered cells to express doxycycline-inducible SAMHD1 variants including WT (SAMWT), catalytically-dead mutant SAMHD1 (SAMMT) or empty vector (MOCK). Comparison of ISGs expression in each group indicated that, relative to MOCK, SAMWT but not SAMMT downregulated ISGs, suggesting that blocking SAMHD1 catalysis promotes innate immunity. To identify SAMHD1 inhibitors, we conducted a structure-based virtual screen of 8,000,000 compounds (Molport). We then assessed the top 500 candidates using cell-based phenotypic screening based on cytarabine sensitization, namely, the inhibitor should sensitize SAMWT but not SAMMT cells to Ara-C toxicity (F). We then selected the 20 best candidates to evaluate for inhibition of SAMHD1 catalysis and chose Samin27 as the most potent lead compound. Importantly, we confirmed direct Samin27 interaction with SAMHD1 protein by nuclear magnetic resonance (NMR) (G). Samin27 treatment of SAMHD1-proficient human and mouse AML lines significantly upregulated ISGs expression. These results suggest that SAMHD1 plays an oncogenic role in AML by suppressing innate immunity. Further analysis is required to determine whether our compound can ablate AML in vivo when combined with currently available immune checkpoint inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hoxa9/meis1-transgenic zebrafish develops acute myeloid leukaemia-like disease with rapid onset and high penetrance.

HOXA9 and MEIS1 are co-expressed in over 50% of acute myeloid leukaemia (AML) and play essential roles in leukaemogenesis, but the mechanisms involved are poorly understood. Diverse animal models offer valuable tools to recapitulate different aspects of AML and link in vitro studies to clinical trials. We generated a double transgenic zebrafish that enables hoxa9 overexpression in blood cells under the draculin (drl) regulatory element and an inducible expression of meis1 through a heat shock promoter. After induction, Tg(drl:hoxa9;hsp70:meis1) embryos developed a preleukaemic state with reduced myeloid and erythroid differentiation coupled with the poor production of haematopoietic stem cells and myeloid progenitors. Importantly, most adult Tg(drl:hoxa9;hsp70:meis1) fish at 3 months old showed abundant accumulations of immature myeloid precursors, interrupted differentiation and anaemia in the kidney marrow, and infiltration of myeloid precursors in peripheral blood, resembling human AML. Genome-wide transcriptional analysis also confirmed AML transformation by the transgene. Moreover, the dihydroorotate dehydrogenase (DHODH) inhibitor that reduces leukaemogenesis in mammals effectively restored haematopoiesis in Tg(drl:hoxa9;hsp70:meis1) embryos and improved their late survival. Thus, Tg(drl:hoxa9;hsp70:meis1) zebrafish is a rapid-onset high-penetrance AML-like disease model, which provides a novel tool to harness the unique advantages of zebrafish for mechanistic studies and drug screening against HOXA9/MEIS1 overexpressed high-risk AML.

MiR-19b-3p promotes tumor progression of non-small cell lung cancer via downregulating HOXA9 and predicts poor prognosis in patients.

MiR-19b-3p has been reported in several types of human cancer. Nevertheless, the expression profile and biological functions of miR-19b-3p remain unclear in non-small cell lung cancer (NSCLC). The expression level of miR-19b-3p was evaluated in NSCLC tissues and cell lines using qRT-PCR. Survival analysis was performed using Kaplan-Meier curves, while the prognostic significance of miR-19b-3p was analyzed using Cox regression analysis in 80 NSCLC patients. The effects of miR-19b-3p on cell proliferation and invasion capacities were analyzed using CCK-8, crystal violet, and transwell assays. Target genes of miR-19b-3p were assessed using luciferase reporter assay, qRT-PCR, Western blot and rescue experiments. MiR-19b-3p was found to be upregulated in human NSCLC tissues and cell lines. The expression of miR-19b-3p was observed to be closely associated with TNM stage and metastasis. High expression of miR-19b-3p was found to be capable of predicting poor clinical prognosis in NSCLC patients. Whilst overexpression of miR-19b-3p was demonstrated to promote the proliferation and invasion of NSCLC cells, knockdown of miR-19b-3p showed an opposite inhibitory effect. Bioinformatics analysis and luciferase reporter assays confirmed that HOXA9 is a direct target of miR-19b-3p. Functional assays demonstrated that NSCLC cell proliferation and invasion were promoted by miR-19b-3p via negative regulation of HOXA9. Finally, overexpression of HOXA9 was shown to partially reverse the tumor promoting effect of miR-19b-3p. This study indicates that miR-19b-3p is a crucial prognostic biomarker of NSCLC, and that targeting of the miR-19b-3p/HOXA9 axis may be a promising strategy in NSCLC therapy.