HMGA2 Research Articles

Telomere as a Therapeutic Target in Dedifferentiated Liposarcoma.

Simple SummaryDDLPS is a non-lipogenic tumor with aggressive clinical behavior. DDLPS patients have limited therapeutic options, especially those with retroperitoneal tumors, and their outcome remains largely unsatisfactory, highlighting the need for novel treatment strategies. Gene expression analysis carried out in clinical samples from primary retroperitoneal DDLPS indicated that these tumors highly express genes involved in telomere maintenance, the cellular process that guarantees immortality to tumor cells. In this study, we evaluated the effect of RHPS4, a molecule able to alter telomere by binding to telomeric structures called G-quadruplexes, in patient-derived DDLPS cell lines. Exposure to RHPS4 induced DNA damage and decreased cell proliferation and migration, thus suggesting telomere as a novel target and G-quadruplex binders as innovative therapeutic agents in DDLPS.Background: Well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS) accounts for ~60% of retroperitoneal sarcomas. WDLPS and DDLPS divergently evolve from a common precursor and are both marked by the amplification of the 12q13–q15 region, leading to the abnormal expression of MDM2, CDK4, and HMGA2 genes. DDLPS is a non-lipogenic disease associated with aggressive clinical behavior. Patients have limited therapeutic options, especially for advanced disease, and their outcome remains largely unsatisfactory. This evidence underlines the need for identifying and validating DDLPS-specific actionable targets to design novel biology-driven therapies. Methods: Following gene expression profiling of DDLPS clinical specimens, we observed the up-regulation of “telomere maintenance” (TMM) pathways in paired DD and WD components of DDLPS. Considering the relevance of TMM for LPS onset and progression, the activity of a telomeric G-quadruplex binder (RHPS4) was assessed in DDLPS patient-derived cell lines. Results: Equitoxic concentrations of RHPS4 in DDLPS cells altered telomeric c-circle levels, induced DNA damage, and resulted in the accumulation of γ-H2AX-stained micronuclei. This evidence was paralleled by an RHPS4-mediated reduction of in vitro cell migration and induction of apoptosis/autophagy. Conclusions: Our findings support telomere as an intriguing therapeutic target in DDLPS and suggest G-quadruplex binders as innovative therapeutic agents.

Development of Primary Monolayer Cell Model and Organotypic Model of Uterine Leiomyoma.

Cellular technologies are one of the most promising areas of biomedicine, which is based on the isolation of cells of various types, followed by their cultivation and use, or the use of their metabolic products, for medical purposes. Today, a significant part of biomedical research is carried out in vitro. On the other hand, organotypic culture can be used as a powerful model system and can complement cell culture and in vivo studies in different biomedical applications. Uterine leiomyoma (UL) is a very common benign tumor and often leads to many reproductive complications. Herein we describe a fast and reliable method of isolation and UL primary cells culturing along with the development of a UL organotypic model. We propose the usage of UL primary cells in experimental work at a first passage to prevent loss of driver mutations in MED12 and HMGA2 genes. New optimized conditions for the growth and maintenance of 2D and 3D models of uterine leiomyoma in vitro are suggested.

Abstract PO-140: Unsupervised clustering and data modeling reveals molecular signatures linked a distinct African American enriched cluster with higher probability of death in triple negative breast cancer

Abstract Triple-negative breast cancer is considered the most aggressive of all breast cancers, with the worst prognosis and increased risk in African Americans. This study aims to characterize proteomic, transcriptomic, and genomic signatures using tissue microarrays from resected TNBC tumors and correlate them with clinical data in an integrated approach to query immune mechanisms and predict patient survival. To this end, our cohort consisted of 118 localized, non-metastatic, resected TNBC patients with diverse demographics (African American 23% (n=28), Hispanic/Latino 9% (n=11), White/Caucasian 36% (n=43) and Other 30%(n=36)) with a median follow-up of 85 months. Here, we studied the patient transcriptomic profiles generated using NanoString sequencing. Immune cell subsets within the tumor microenvironment were assessed on tumor microarray slides by Multiplex Immunohistochemistry Consecutive on Single Slide (MICSSS). Protein markers measured include PD-L1, CD3, and CD8, CD20, CD66b, FOXP3, DC-LAMP, TLS, CD68, and CD163. We utilized unsupervised clustering of the patient transcriptomes as a data-driven approach to identify patient clusters defined by co-expressed RNA molecules. Then, we performed differential expression using mixed linear models for both data types (RNA & Protein), to compare these clusters to each other identifying cluster-specific molecular signatures and association to outcomes. This unbiased modeling strategy enabled us to include critical covariates in the analysis such as demographics, neoadjuvant treatment, clinical tumor parameters, and quantify the effect of potential confounders. Finally, these results revealed a cluster of patients with the best survival associated with high expression of genes such as GZMK/A, CCR5, IL10RA, IL2RG, and the highest levels of CD8, PD-L1, CD163, FOXP3, and CD68 protein tissue markers. By contrast, the cluster with the worst outcomes was enriched in African Americans (44% n=8/18, p.value<0.05, compared to an average of 16% (n=8/25) in other clusters). Furthermore, this cluster included increased BRCA1, MYC, KIF2C, BIRC5, and HMGA1 gene expression, with an absence of immune proteomic markers, and it was independent of chemotherapy type, histology scores, tumor status, or lymphatic vessel invasion. Genomic alteration analyses are pending. In conclusion, we are using a data-driven approach to characterize patients with triple-negative breast cancer and patient clusters by integrating proteomic and transcriptomic molecular tumor profiles, and we identified a gene cluster enriched in African Americans associated with worse outcomes and poor immune infiltration. By combining a wider immune tissue characterization using an extended panel of MICSSS markers together with genetic mutational and expression profiling of tumors, we expect to refine and distinguish subsets of high-risk TNBC patients for whom more aggressive tailored treatment regimens may be indicated. Citation Format: Edgar Gonzalez-Kozlova, Clelia Chalumeau, Ilaria LaFace, Charles Shapiro, Guray Akturk, Diane Marie Del Valle, Sacha Gnjatic. Unsupervised clustering and data modeling reveals molecular signatures linked a distinct African American enriched cluster with higher probability of death in triple negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-140.

A Rare Case of Myoid Hamartoma Masquerading As Invasive Breast Carcinoma

Breast Myoid Hamartoma (MH) is a rare type of neoplasm with a poorly understood pathogenesis. Very few literatures have reported such disease with an unclear prognosis and malignant potentiality. Some isolated studies have shown that breast Myoid Hamartoma (MH) may be genetically related to other types of tumours with the involvement of HMGA2 gene. We reported a case of a 64-year-old post-menopausal lady with an underlying chronic idiopathic axonal polyneuropathy (CIAP) that was referred to our centre for a suspected right breast tumour. Clinical and imaging proved the disease to be malignant, however, core biopsy results showed otherwise. Ultrasound of the right breast showed a solid mass with a hypoechoic heterogeneous echotexture and posterior shadowing. A Mammogram highlighted a dense lesion in the right breast with radiolucent halo and macrocalcification. It was reported as BIRADS 4 category. Managing breast Myoid Hamartoma (MH) is proved to be of great challenge to clinicians as meticulous clinical acumen is needed to strategize a proper plan and management, most importantly, not to overlook the disease as the malignant transformation has been reported before.

High mobility group AT-hook 2 regulates osteoblast differentiation and facial bone development

The mutation and deletion of high mobility group AT-hook 2 (Hmga2) gene exhibit skeletal malformation, but almost nothing is known about the mechanism. This study examined morphological anomaly of facial bone in Hmga2−/− mice and osteoblast differentiation of pre-osteoblast MC3T3-E1 cells with Hmga2 gene knockout (A2KO). Hmga2−/− mice showed the size reduction of anterior frontal part of facial bones. Hmga2 protein and mRNA were expressed in mesenchymal cells at ossification area of nasal bone. A2KO cells differentiation into osteoblasts after reaching the proliferation plateau was strongly suppressed by alizarin red and alkaline phosphatase staining analyses. Expression of osteoblast-related genes, especially Osterix, was down-regulated in A2KO cells. These results demonstrate a close association of Hmga2 with osteoblast differentiation of mesenchymal cells and bone growth. Although future studies are needed, the present study suggests an involvement of Hmga2 in osteoblast-genesis and bone growth.

Genome analyses revealed genetic admixture and selection signatures in Bos indicus

The genomic diversity and relationship among seven diverse cattle breeds viz. Sahiwal, Tharparkar, Gir, Vechur, Ongole, Kangayam and Hariana were investigated in 132 random samples based on high density SNP array comprising > 777 K SNPs. A total of 1993 SNPs (0.25% of the total) having greater power (FST ≥ 0.20) to differentiate these cattle populations were identified, and utilized to partition genome of each animal into a predefined number of clusters. The structure of these cattle indicated shared ancestry of dairy breeds viz. Gir, Tharparkar and Sahiwal. Most of the animals (> 76%) of different populations under study except Vechur clustered into their own group of animals called breed. Vechur population retained highest rate of admixture, consistent with its crossing with other breeds. Ongole, Kangayam and Hariana shared comparatively less of their genome (≤ 15%) with other breeds. The study indicated that all seven breeds evolved from their independent ancestry but there was intermixing of these breeds in the recent past. The selection signatures identified between draft (Kangayam) and dairy breeds included several genes like FAM19A2, RAB31P, BEST3, DGKA, AHCY, PIGU and PFKP which are involved in immune response, metabolic pathway, transportation of glucose and sugars, signaling pathways, cellular processes, cell division and glycolysis regulation, respectively. Moreover, these genomic regions also harbour QTLs affecting milk performance traits. The signatures were also identified even between the dairy breeds. In comparison to large-sized cattle, there were significant differences in the number of QTLs affecting production (body weight, growth rate etc.) and morphological traits (height) in short-statured Vechur breed. The presence of HMGA2 gene in the selection signature on chromosome 5 may explain the variations in stature between these cattle.

KIFC1 promotes aerobic glycolysis in endometrial cancer cells by regulating the c-myc pathway.

Endometrial cancer (EC) is a common gynecological malignant tumor worldwide. It is imperative to study pathogenesis and therapeutic targets for improving the prognosis of EC. The present study aimed to explore the function and mechanism of kinesin family member C1 (KIFC1) in EC. EC tumor and adjacent normal tissues were collected from 68 pairs of patients. The expression of KIFC1 in tissues and EC cells was analyzed by immunohistochemistry, qRT-PCR or western blot. MTT assay was used to test the cell viability. Flow cytometry was used to determine apoptosis and the cell cycle. Glucose uptake, lactate production, ATP contents and lactate dehydrogenase (LDH) activity were evaluated by a glucose metabolism kit. The expression of HMGA1, c-myc and glycolytic genes was assessed using western blot or qRT-PCR. A mouse xenograft model was established in BALB/c mice to detect tumor growth in vivo. KIFC1 was significantly upregulated in EC tumor tissues compared to adjacent normal control tissues. The upregulated expression of KIFC1 was correlated with poor prognosis in patients. Lentiviral-mediated overexpression of KIFC1 observably enhanced cell viability and reduced the apoptotic rate of Ishikawa and HEC-1B cells. Cell cycle progression was also expedited in the KIFC1 vector group. Moreover, overexpression of KIFC1 elevated glucose uptake, lactate production, ATP contents and LDH activity. However, knockdown of KIFC1 by short hairpin RNA (shRNA) showed the reverse effect on cellular functions. In addition, the expression of c-myc, GLUT1, LDHA and HK2 was increased by the KIFC1 vector. Moreover, HMGA1 regulated the expression of c-myc and glycolytic genes. Upregulated HMGA1 could rescue the effect of KIFC1 knockdown on cellular functions and the expression of glycolytic genes. Finally, KIFC1 knockdown inhibits tumor growth in vivo. The upregulation of KIFC1 was correlated with poor prognosis in EC. KIFC1 promoted aerobic glycolysis in endometrial cancer cells by regulating the HMGA1/c-myc pathway. KIFC1 may be a potential target for the diagnosis and therapy of EC.

Restoration of MiR-34a Expression by 5-Azacytidine Augments Alimta -Induced Cell Death in Non-Small Lung Cancer Cells by Downregulation of HMG B1, A2 and Bcl-2 Pathway.

Alimta (Pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. The aim of the present study was to investigate the combination effect of 5-Azacytidine (5-aza) and Alimta on the miR-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer A549 cells. In this experimental study, lung cancer A549 cells were treated with various concentrations of Alimta alone and combined with 5-Aza. Then, viability was assessed by trypan blue and MTT assays. mRNA expressions were performed by real time-polymerase chain reaction (PCR) and western blot. Flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest. Alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 12 μM. Pretreatment of the cells with 5-aza (5 μM) induced a synergistic cytotoxic effect with IC50 of 3 μM. Sequential exposure of the cells to 5-aza and Alimta enhanced miR-34a expression and significantly downregulated HMGB1, HMGA2 and BCL-2 expressions. Also, it was associated with reduction of nuclear HMGB1 and HMGA2 content. Caspase-3 activation, HMGB1 release into extracellular space and staining of the cells with annexine V/PI suggested that 5-aza reduced late apoptotic/necrotic cell death induced by Alimta. In addition, combination of 5-aza and Alimta arrested the cells at S and sub-G1 phases and inhibited colony formation. 5-aza synergistically enhances Alimta induced apoptotic cell death through HMG proteins regulation, MIR34A gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.

Restoration of MiR-34a Expression by 5-Azacytidine Augments Alimta -Induced Cell Death in Non-Small Lung Cancer Cells by Downregulation of HMG B1, A2 and Bcl-2 Pathway

Objective Alimta (Pemetrexed) as an antifolate drug has been approved for the treatment of lung cancer. The aim of the present study was to investigate the combination effect of 5-Azacytidine (5-aza) and Alimta on the miR-34a and its target genes expression and induction of apoptotic cell death in non-small lung cancer A549 cells. Materials and Methods In this experimental study, lung cancer A549 cells were treated with various concentrations of Alimta alone and combined with 5-Aza. Then, viability was assessed by trypan blue and MTT assays. mRNA expressions were performed by real time-polymerase chain reaction (PCR) and western blot. Flow cytometry used to detect apoptotic/ necrotic cells and cell cycle arrest. Results Alimta alone reduced viability of the cells in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 12 µM. Pretreatment of the cells with 5-aza (5 µM) induced a synergistic cytotoxic effect with IC50of 3 µM. Sequential exposure of the cells to 5-aza and Alimta enhanced miR-34a expression and significantly downregulated HMGB1, HMGA2 and BCL-2 expressions. Also, it was associated with reduction of nuclear HMGB1 and HMGA2 content. Caspase-3 activation, HMGB1 release into extracellular space and staining of the cells with annexine V/PI suggested that 5-aza reduced late apoptotic/necrotic cell death induced by Alimta. In addition, combination of 5-aza and Alimta arrested the cells at S and sub-G1 phases and inhibited colony formation. Conclusion5-aza synergistically enhances Alimta induced apoptotic cell death through HMG proteins regulation, MIR34A gene expression and intrinsic apoptosis mechanism, providing a promising combination therapy in clinical lung cancer therapy.

Critical role of the high mobility group A proteins in hematological malignancies.

The high mobility group A (HMGA) protein family is composed of three non‐histone chromatin remodeling proteins that act as architectural transcriptional factors. Indeed, although HMGA proteins lack transcriptional activity per se, they bind the minor groove of DNA at AT‐rich sequences, and, interacting with the transcription machinery, are able to modify chromatin modeling, thus regulating the expression of several genes. HMGA proteins have been deeply involved in embryogenesis process, and a large volume of studies has pointed out their key role in human cancer. Here, we review the studies on the role of the HMGA proteins in human hematological malignancies: they are overexpressed in most of the cases and their expression correlates with a reduced survival. In some cases, such as in acute lymphoblastic leukemia and acute myelogenous leukemia, HMGA2 gene rearrangements have been also described. Finally, recent studies evidence a synergism between HMGA and EZH2 in diffuse B‐cell lymphomas, suggesting an innovative therapy for this disease based on the inhibition of the function of both these proteins.

Uterine cellular leiomyomas are characterized by common HMGA2 aberrations, followed by chromosome 1p deletion and MED12 mutation: morphological, molecular, and immunohistochemical study of 52 cases.

Cellular leiomyoma (CL) represents an uncommon variant of uterine leiomyoma with limited data concerning its immunohistochemical and molecular profile. We performed a comprehensive analysis of 52 CL cases all of which were analyzed immunohistochemically. Molecular analysis was possible in 32 cases with sufficient DNA, and 38 cases with sufficient RNA. The immunohistochemical results showed a high expression of smooth muscle markers (calponin (100%), desmin (100%), smooth muscle actin (98.1%), caldesmon (96.1%), transgelin (96.1%), smooth muscle myosin heavy chain (86.5%), and smoothelin (61.5%)). Concerning markers of endometrial stromal differentiation, the expression of CD10 was observed in 65.4% cases (42.2% with H-score > 50), and IFITM1 in 36.5% cases (1.9% with H-score > 50). 36.5% showed HMGA2 overexpression at the IHC level, associated with increased mRNA expression in 14/14 cases. The rearrangement of the HMGA2 gene was detected in 13.2%. Chromosome 1p deletion was found in 19.3%, while 9.4% of tumors showed a pathogenic mutation in the MED12 gene. In conclusion, CL is immunohistochemically characterized by a high expression of "smooth muscle" markers commonly associated with a co-expression of "endometrial stromal" markers, where IFITM1 shows superior performance compared to CD10 regarding its specificity for differentiation from endometrial stromal tumors. The sensitivity of smoothelin in CL seems rather low, but no data is available to assess its specificity. On a molecular level, the most common mutually exclusive aberration in CL affects HMGA2, followed by chromosome 1p deletions and MED12 mutations.

Some pleomorphic adenomas of the breast share PLAG1 rearrangements with the analogous tumour of the salivary glands

Pleomorphic adenoma (PA) of the breast, and especially its malignant transformation, is extremely rare and represents a diagnostic pitfall. Molecular alterations in this entity have not been investigated. We aimed to examine the clinicopathological features of our breast PAs and perform molecular analysis. Seven cases of breast PA, including two cases of carcinoma ex PA, were analysed. PLAG1 and HMGA2 gene rearrangements were assayed by fluorescence in-situ hybridisation (FISH) and RNA sequencing (RNA-Seq), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to verify RNA sequencing results. All seven cases of breast PA occurred in women. The histological features were similar to the analogous tumour in salivary glands, including a dual epithelial-myoepithelial component and negativity of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) by immunohistochemistry. Of the two cases with carcinoma ex PA, one demonstrated minimal invasion and one was extensively invasive. PLAG1 rearrangements were identified in two cases (28.6%), but no rearrangements of HMG2A were found. A novel fusion product in PAs, TRPS1-PLAG1, was identified in one case. No patients had recurrence or metastasis with a follow-up period of 6-158 months. Breast PA is rare, but it is an important differential diagnosis of breast pathology with the potential to develop carcinoma ex PA. We report a novel TRPS1-PLAG1 fusion gene in breast PA.

HMGA1 promotes hepatocellular carcinoma proliferation, migration, and regulates cell cycle via miR-195-5p.

HMGA1 has been reported to be aberrantly expressed and correlate with the poor prognosis of many carcinomas. This study aimed to investigate the clinical significance and molecular mechanism of HMGA1 as a tumor-suppressing gene in hepatocellular carcinoma (HCC). Analysis of TCGA dataset by TANRIC website and R2 platform, we found that HMGA1 expression was significantly higher in HCC tissues compared to that in normal liver tissues and was associated with Edmondson grade. Patients with highly expressed HMGA1 had worse overall survival. Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes analysis showed the potential relationships between HMGA1 and other genes in HCC. We also demonstrated that the downregulation of HMGA1 dramatically suppressed the proliferation and migration of HCC cells. Furthermore, ectopic expression of HMGA1 blocked G0/G1 to S transition. Subsequent investigation characterized HMGA1 as a direct target of miR-195-5p, and miR-195-5p downregulation abrogated the effect of HMGA1 on HCC proliferation, migration, and cell cycle arrest. In addition, we also demonstrated that miR-195-5p downregulation abrogated the effect of HMGA1 on HCC growth in vivo. Taken together, our data provide strong evidence that HMGA1 promotes HCC and is negatively regulated by the tumor-suppressor, miR-195-5p.

HMGA2 rs968697 T > C polymorphism is associated with the risk of colorectal cancer

A genetic polymorphism (rs968697 T > C) in the HMGA2 gene has recently been linked to an increased risk of hepatoblastoma. However, no studies have been conducted to investigate the effect of the polymorphism on the risk of colorectal cancer (CRC). The study aimed to explore whether the rs968697 polymorphism had a significant impact on CRC risk. A total of 500 CRC patients and 500 age and gender matched healthy individuals were genotyped by using the SNaPshot method. Quantitative real-time PCR technology was used to detect the relative expression of the HMGA2 gene in 30 pairs of primary CRC and adjacent non-cancerous tissues. Results: HMGA2 rs968697 polymorphism was significantly associated with CRC risk [CC vs. TT: OR = 0.20, 95%CI = 0.06–0.70, P = 0.01; (CC + CT) vs. TT: OR = 0.71, 95%CI = 0.53–0.96, P = 0.02; CC vs. (CT + TT): OR = 0.21, 95%CI = 0.06–0.73, P = 0.01; C vs. T: OR = 0.67, 95%CI = 0.51–0.89, P < 0.01]. The analysis based on tumor stage indicated that the CRC patients with HMGA2 rs968697 C allele were less likely to have high-stage tumors. Furthermore, the genotype-tissue expression analysis revealed that the rs968697 CC genotype was linked to the low expression of HMGA2 gene. The in silico analysis revealed that the rs968697 polymorphism in the promoter region of the HMGA2 gene could influence transcription factor binding, including ATF6, DBP, CDPCR3, DR3, NRSF, PAX8, PPARA, SZF11, TAXCREB and POLR2A. In conclusion, our findings suggested that the HMGA2 rs968697 polymorphism was linked to CRC risk and could be used as a biomarker to detect CRC risk.

Abstract 625: Co-amplification of CDK4 and MDM2 plus HMGA2 fusion in a patient with myogenic differentiation dedifferentiated liposarcoma

Abstract BACKGROUND: Dedifferentiated liposarcoma (DDLPS) with myogenic differentiation is a rare subtype of soft tissue sarcoma, and its pathogenesis remains unclear. The morphological resemblance with myofibroblastic sarcoma and rhabdomyosarcoma may easily result in misdiagnosis. Some studies have shown that myogenic differentiation is considered as a poor prognostic factor for DDLPS; consequently, it is of great clinical significance for its accurate diagnosis. Next-generation sequencing (NGS) can be performed to better explore the molecular characteristics and targeted therapies of myogenic differentiation DDLPS. Here, we reported a rare case of myogenic differentiation DDLPS harboring co-amplification of CDK4 and MDM2 plus HMGA2 gene fusion, which was clarified via NGS. CASE PRESENTATION: A 61-year-old male patient presented with local soft tissue thickening in the right groin and scrotum by PET-CT. Pathological examination revealed a 5.8*4.5*3.5cm mass in the right groin with an enveloping appearance and gray-brown section. Microscopic examination showed the parenchymal mass was composed of spindle cells. Immunohistochemistry (IHC) reported that spindle cells were diffusely positive for desmin and vimentin; focally positive for epithelial membrane antigen, smooth muscle actin and actin; and negative for ALK, S-100, myogenin, CD30, CK, h-caldesmon, MyoD1, STAT6, SOX-10 and β-catenin. The approximate Ki-67 label index was 40%. Combining with the morphology and IHC, the patient was diagnosed with myogenic differentiated soft tissue sarcoma. After surgical resection, NGS was performed on resected tissues to further identify the subtype due to its high aggressiveness. Fresh surgical tissues were tested by the NGS-based 825-gene DNA panel and 395-gene RNA panel. The tumor samples contained three somatic mutations: ELAC2 p.H194R (14.4%), HRH2 p.S325F (18.1%), and KEL p.G167D (19.3%). Copy number analysis revealed copy number gains for CDK4, MDM2 and IGF1R in the patient. He also carried HMGA2(E3)-CCDC91(E2), FRS2(E3)-BCAT1(E3) and ATG5(E5)-DSG3(E13) gene fusion. CDK4 and MDM2 gene amplification is the clinical diagnostic marker of DDLPS, this patient was finally diagnosed as DDLPS with myogenic differentiation. Additionally, HMGA2 gene fusion was firstly reported in myogenic differentiation DDLPS, which was often discussed in lipoma before. CONCLUSION: To date, limited scientific literature is available for a comprehensive understanding of the genetic characteristics of myogenic differentiation DDLPS. For the first time, we reported a case of myogenic differentiation DDLPS with simultaneous CDK4, MDM2 amplification and HMGA2 gene fusion, which may be the potential tumorigenic mechanism of myogenic differentiation DDLPS. Citation Format: Gang Huang, Xianan Li, Chunyang Wang, Qifan He, Xiaojuan Wang. Co-amplification of CDK4 and MDM2 plus HMGA2 fusion in a patient with myogenic differentiation dedifferentiated liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 625.

HMGA2 Gene Expression Profile Improves Risk Stratification in Acute Myeloid Leukemia

Abstract Introduction: In adult acute myeloid leukemia (AML), clinical outcome is mainly predicted by age, cytogenetics and specific gene mutations (Papaemmanuil et al ., N. Engl. J. Med., 2016; Dohner et al., Blood, 2017). The 2017 European LeukemiaNet (ELN) expert panel for AML diagnosis recommends screening for mutations in NPM1, CEBPA, RUNX1, FLT3, TP53, and ASXL1 genes inaddition to chromosomal anomalies (Dohner et al., Blood, 2017). The genetic and cytogenetic risk stratification guides AML therapy, but it remains insufficient to select the optimal therapy especially in the intermediate genetic risk category. Several gene expression signatures have been proposed to further refine AML risk stratification (Li et al ., J. Clin. Oncol., 2013; Ng et al ., Nature, 2016). However, they have not been widely adopted because of technical challenges in implementing large gene signatures in clinical settings. Accurate biomarkers are still needed to improve prognostic assessment in AML. Methods: We analyzed RNA sequencing data of 430 clinically annotated AML samples and identified HMGA2 asa new candidate prognostic marker. A RT-qPCR test was developed and validated in order to facilitate implementation in clinical laboratories. The association of HMGA2 expression with complete remission (CR), overall survival (OS), relapse-free survival (RFS) and cumulative incidence of relapse (CIR), was studied in a training cohort of 358 diagnostic AML samples from intensively treated patients. Multivariable models were adjusted for age, white blood cell counts, cytogenetic risk, NPM1 and FLT3 -ITD mutations and, except for CR prediction, hematopoietic stem cell transplantation as a time-dependent variable. The prognostic value of the HMGA2 test was validated in an independent cohort of 260 diagnostic AML samples from patients enrolled in the UK National Cancer Research Institute (NCRI) AML17 trial. Results: In the training cohort, HMGA2 was highly expressed in 22.3% of AML, mostly in patients with intermediate or adverse cytogenetics. A high expression level of HMGA2 (H+) was associated with lower CR frequency (58.8% vs 83.4%, p Next, we investigated whether the HMGA2 test could improve prognostic assessment in AML patients classified according to the 2017 ELN genetic risk stratification (Dohner et al., Blood, 2017). We observed that a positive HMGA2 test identified 45 of 87 (51.7%) ELN adverse risk patients who were unresponsive to current treatments. Importantly, the HMGA2 test was also positive in 43.5% of intermediate risk patients who were negative for NPM1, FLT3 -ITD and biallelic CEBPA mutations, and in 66.7% of patients with TP53, RUNX1 or ASXL1 mutations. This finding is clinically relevant, especially if screening for TP53, RUNX1 or ASXL1 mutations is not available. Consistent with results in the training cohort, H+ was a good predictor of poor survival and a higher CIR in the validation cohort (Fig. C-D). Multivariable analyses also confirmed that H+ was strongly and independently associated with a higher risk of primary refractory disease (CR: aOR = 3.98 [1.36-11.65], p= 0.01), lower OS (aHR = 2.03 [1.36-3.03], p Conclusion: High HMGA2 expression adds significant independent prognostic value to known clinical and genetic prognostic factors in AML, and is predictive of poor clinical outcomes with standard AML therapies. The HMGA2 test can complement the current AML tests to improve treatment orientation and be integrated in ongoing and future prospective clinical trials studying innovative therapies to increase survival of HMGA2 positive AML patients. Download : Download high-res image (149KB) Download : Download full-size image Figure 1 . Disclosures Wei: Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding. Fleming: Abbvie: Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; MSD: Speakers Bureau. Schwarer: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Specialised Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sauvageau: ExCellThera: Other: Chief Executive Officer and Chief Scientific Officer.

Optimization of CUT&Tag product recovery and library construction method.

The emerging cleavage under target and tagment (CUT&Tag) technology uses Tn5 transposase to cleavage near the DNA binding site of target protein and study the generated DNA fragments by the next-generation sequencing. It can quickly identify protein-DNA interactions, which greatly simplifies the experimental process of ChIP-Seq. After CUT&Tag tagment reaction, DNA recovery or other post-processing is required to perform library construction PCR. Different recovery methods have significant impact respectively. By establishing Streptavidin beads recovery CUT&Tag(srCUT&Tag), we can quickly and conveniently complete the product recovery of CUT&Tag. We carried out CUT&Tag assay of H3K4me3, RNA Polymerase II (RNA polymerase II, RNAPII), transcription factor CTCF and HMGA1 in K562 cells with different recovery methods, including ethanol precipitation, fragment separation magnetic beads (SPRI) Magnetic bead recovery, direct PCR method, as well as our srCUT&Tag recovery method. The results show that among the CUT&Tag results of four different targets, the SPRI magnetic bead recovery and our srCUT&Tag methods have higher recovery efficiency than the direct PCR method and ethanol precipitation method. All CUT&Tag results showed that the recovery of SPRI magnetic beads would lose most of the product fragments less than 150 bp. In the recovery of CTCF and HMGA1, direct PCR lost most of the fragments larger than 300 bp and has significant difference from result of other recovery method. This enables srCUT&Tag to provide more real and higher-resolution information than other recovery method. In summary, the newly established srCUT&Tag recovery method can improve the efficiency of CUT&Tag library construction and obtain better data quality compared with the existing CUT&Tag product recovery method, providing a better technical choice for epigenetics research.

Glycine alleviates fluoride‐induced oxidative stress, apoptosis and senescence in a porcine testicular Sertoli cell line

Glycine is a well-known free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. Excessive fluoride exposure is associated with multiple types of cellular damage in humans and animals. The objective of the present study was to investigate the protective effects of glycine on sodium fluoride (NaF) exposure and the possible underlying mechanisms in a porcine testicular Sertoli cell line model. Cellular viability and proliferation were examined following NaF exposure and glycine supplementation, and glycine dramatically ameliorated the decreases in NaF-induced porcine testicular Sertoli cell viability and proliferation. Further investigations revealed that glycine decreased NaF-induced intracellular reactive oxygen species production, DNA fragment accumulation and the apoptosis incidence in the porcine testicular Sertoli cell line; in addition, glycine improved mitochondrial function and ATP production. Notably, results of the SPiDER-β-Gal analysis suggested that glycine alleviated NaF-induced cellular senescence and downregulated P53, P21, HMGA2 and P16INK4a gene expression in the porcine testicular Sertoli cell line. Collectively, the beneficial effects of glycine alleviate NaF-induced oxidative stress, apoptosis and senescence, and together with our previous findings, support the hypothesis that glycine plays an important role in protecting against NaF exposure-induced impairments in the porcine testicular Sertoli cell line.

Deregulation of a Cis-Acting lncRNA in Non-small Cell Lung Cancer May Control HMGA1 Expression.

BackgroundLong non-coding RNAs (lncRNAs) have long been implicated in cancer-associated phenotypes. Recently, a class of lncRNAs, known as cis-acting, have been shown to regulate the expression of neighboring protein-coding genes and may represent undiscovered therapeutic action points. The chromatin architecture modification gene HMGA1 has recently been described to be aberrantly expressed in lung adenocarcinoma (LUAD). However, the mechanisms mediating the expression of HMGA1 in LUAD remain unknown. Here we investigate the deregulation of a putative cis-acting lncRNA in LUAD, and its effect on the oncogene HMGA1.MethodsLncRNA expression was determined from RNA-sequencing data of tumor and matched non-malignant tissues from 36 LUAD patients. Transcripts with significantly deregulated expression were identified and validated in a secondary LUAD RNA-seq dataset (TCGA). SiRNA-mediated knockdown of a candidate cis-acting lncRNA was performed in BEAS-2B cells. Quantitative real-time PCR was used to observe the effects of lncRNA knockdown on the expression of HMGA1.ResultsWe identified the lncRNA RP11.513I15.6, which we refer to as HMGA1-lnc, neighboring HMGA1 to be significantly downregulated in both LUAD cohorts. Conversely, we found HMGA1 significantly overexpressed in LUAD and anticorrelated with HMGA1-lnc. In vitro experiments demonstrated siRNA-mediated inhibition of HMGA1-lnc in immortalized non-malignant lung epithelial cells resulted in a significant increase in HMGA1 gene expression.ConclusionOur results suggest that HMGA1-lnc is a novel cis-acting lncRNA that negatively regulates HMGA1 gene expression in lung cells. Further characterization of this regulatory mechanism may advance our understanding of the maintenance of lung cancer phenotypes and uncover a novel therapeutic intervention point for tumors driven by HMGA1.

Not chronic exercise but acute exercise is related with increased cell survival, enhanced cell proliferation and decreased cell apoptosis

Abstract Background Endurance training can be described as long-time activity characterized by high dynamic and low to high power and was proven beneficial in CVD prevention. Nevertheless, studies reported that excessive endurance training can act as double-edged sword inducing the inflammatory response and cell apoptosis. This negative effect can be the reason of exercise duration. Purpose In order to investigate the effect of the training duration we compared and analysed acute vs chronic exercise by using in silico technique and we performed two different bioinformatics analysis: tissue-specific and cardiovascular process specific. Materials Bioinformatics analysis miRNA targets predictions, data filtering and visualization as interaction networks were performed. We used multiMiR 1.4 R package, hemopoiesis, angiogenesis, cardiac muscle functions, muscle hypertrophy process was performed a screening of the GO terms for the presence of the key words using the biomaRt package in R, Gene-gene interaction data were retrieved from String App, Cytoscape. Enrichment analysis of Reactome and KEGG pathways using String App. p&amp;lt;0.05. Results Enrichment analysis of Reactome and Kegg pathways revealed strong regulation of senescence-related pathways, RUNX1 expression and activity, Ca2+ signaling involving AGO genes. For the first time our in silico analysis showed that AGO, HMGA2 genes may be associated with adaptive changes in response to exercise. Importantly, we found, that PI3K/AKT signalling (pathway incusing cell survival, growth, and proliferation) was present only in acute but not in chronic exercise. Conclusions In our analysis long-term training miRNA-gene target interaction was different than the short-term training. As PI3K/AKT signaling was present only in acute exercise, we suggest that not long-term but short-term training can be related with increased cell survival, enhanced cell proliferation, and decreased cell apoptosis. Genes associated cardiac muscle function Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): “Preludium” from the National Science Center, Poland