Epigenetic Priming with Decitabine Augments the Therapeutic Effect of Cisplatin on Triple-Negative Breast Cancer Cells through Induction of Proapoptotic Factor NOXA

Epigenetic alterations caused by aberrant DNA methylation have a crucial role in cancer development, and the DNA-demethylating agent decitabine, is used to treat hematopoietic malignancy. Triple-negative breast cancers (TNBCs) have shown sensitivity to decitabine; however, the underlying mechanism of its anticancer effect and its effectiveness in treating TNBCs are not fully understood. We analyzed the effects of decitabine on nine TNBC cell lines and examined genes associated with its cytotoxic effects. According to the effect of decitabine, we classified the cell lines into cell death (D)-type, growth inhibition (G)-type, and resistant (R)-type. In D-type cells, decitabine induced the expression of apoptotic regulators and, among them, NOXA was functionally involved in decitabine-induced apoptosis. In G-type cells, induction of the cyclin-dependent kinase inhibitor, p21, and cell cycle arrest were observed. Furthermore, decitabine enhanced the cytotoxic effect of cisplatin mediated by NOXA in D-type and G-type cells. In contrast, the sensitivity to cisplatin was high in R-type cells, and no enhancing effect by decitabine was observed. These results indicate that decitabine enhances the proapoptotic effect of cisplatin on TNBC cell lines that are less sensitive to cisplatin, indicating the potential for combination therapy in TNBC.

SORT1/LAMP2-mediated Extracellular Vesicle Secretion and Cell Adhesion Are Linked to Lenalidomide Resistance in Multiple Myeloma

Multiple myeloma (MM) is a hematopoietic malignancy whose prognosis has improved with the development of new agents such as lenalidomide over the last decade. However, long-term exposure to drugs induces the acquisition of resistance by MM cells and leads to treatment failure and poor prognosis. Here, we show the molecular and cellular mechanisms of lenalidomide resistance in MM. In a comparison between lenalidomide-resistant cell lines and the parental cell lines, the EV (Extracellular versicles) secretion and adherence abilities were significantly elevated in the resistant cells. Whole-transcriptome analysis revealed that the SORT1 and LAMP2 genes were key regulators of EV secretion. Silencing of these genes caused decreased EV secretion and loss of cell adhesion in the resistant cells, resulting in increased sensitivity to lenalidomide. Analysis of publicly available transcriptome data confirmed the relationship between genes related to EV secretion and cell adhesion and patient prognosis. Together, our findings reveal a novel mechanism of lenalidomide resistance in MM mediated by EV secretion and cell adhesion via SORT1 and LAMP2.

Case Report: Long-Term Response to Radiotherapy Combined With Targeted Therapy in Histiocytic Sarcoma Harboring Mutations in MAPK and PI3K/AKT Pathways

BackgroundHistiocytic sarcoma (HS) is a rare hematopoietic malignancy with an aggressive clinical presentation associated with a poor overall survival. To date, surgical resection, radiation therapy, and chemotherapy were often utilized for HS, but curative effects are rather disappointing.Case PresentationA 19-year-old female was referred to our hospital with a pathologic diagnosis of HS in December 2017. The patient had a severe airway obstruction resulting from a large mass (6.0 cm × 4.4 cm) arising from the left parapharyngeal space. She did not respond to cyclophosphamide, doxorubicin, vincristine, prednisone, and etoposide (CHOEP) chemotherapy, then she was switched to radiotherapy and crizotinib according to next-generation sequencing (NGS) results (mutations in MET and MAP2K1). The patient got a partial response after radiotherapy and crizotinib, then she switched to imatinib combined with thalidomide treatment. The patient got a long-term complete response from the treatment and is alive 44 months after initial diagnosis without disease progression. Further KEGG pathway enrichment analysis of NGS results from patient’s tissue revealed that phosphatidylinositol 3′ kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK) pathways were activated in this HS patient. We further performed experiments in vitro in a canine histiocytic sarcoma cell line DH82, in order to explore the possible mechanism of imatinib plus thalidomide in HS. Results of cell counting kit-8 (CCK8) assays showed that the proliferation activity of DH82 was significantly inhibited by imatinib but not thalidomide. Combined thalidomide and imatinib treatment did not improve the inhibitory effects of imatinib to DH82. Results of Western blot confirmed the inhibitory effects of imatinib on DH82 by targeting activation of MAPK and PI3K/AKT pathways.ConclusionRadiotherapy combined with targeted therapy guided by NGS may be promising, and further perspective clinical trial is warranted for the localized HS.

USAID Associated with Myeloid Neoplasm and VEXAS Syndrome: Two Differential Diagnoses of Suspected Adult Onset Still’s Disease in Elderly Patients

Background: Patients with solid cancers and hematopoietic malignancy can experience systemic symptoms compatible with adult-onset Still’s disease (AOSD). The newly described VEXAS, associated with somatic UBA1 mutations, exhibits an overlap of clinical and/or biological pictures with auto inflammatory signs and myelodysplastic syndrome (MDS). Objectives: To describe a cohort of patients with signs of undifferentiated systemic autoinflammatory disorder (USAID) concordant with AOSD and MDS/chronic myelomonocytic leukemia (CMML) and the prevalence of VEXAS proposed management and outcome. Methods: A French multicenter retrospective study from the MINHEMON study group also used for other published works with the support of multidisciplinary and complementary networks of physicians and a control group of 104 MDS/CMML. Results: Twenty-six patients were included with a median age at first signs of USAID of 70.5 years with male predominance (4:1). Five patients met the criteria for confirmed AOSD. The most frequent subtypes were MDS with a blast excess (31%) and MDS with multilineage dysplasia (18%). Seven patients presented with acute myeloid leukemia and twelve died during a median follow-up of 2.5 years. Six out of 18 tested patients displayed a somatic UBA1 mutation concordant with VEXAS, including one woman. High-dose corticosteroids led to a response in 13/16 cases and targeted biological therapy alone or in association in 10/12 patients (anakinra, tocilizumab, and infliximab). Azacytidine resulted in complete or partial response in systemic symptoms for 10/12 (83%) patients including 3 VEXAS. Conclusion: Systemic form of VEXAS syndrome can mimic AOSD. The suspicion of USAID or AOSD in older males with atypia should prompt an evaluation of underlying MDS and assessment of somatic UBA1 mutation.

Perturbation of p38α MAPK as a Novel Strategy to Effectively Sensitize Chronic Myeloid Leukemia Cells to Therapeutic BCR-ABL Inhibitors

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5–3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38β, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.

Identification of m6A-Related lncRNAs Associated With Prognoses and Immune Responses in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) remains the most common type of hematopoietic malignancy in adults and has an unfavorable outcome. Herein, we aimed to construct an N6-methylandenosine (m6A)-related long noncoding RNAs (lncRNAs) signature to accurately predict the prognosis of patients with AML using the data downloaded from The Cancer Genome Atlas (TCGA) database.Methods: The RNA-seq and clinical data were obtained from the TCGA AML cohort. First, Pearson correlation analysis was performed to identify the m6A-related lncRNAs. Next, univariate Cox regression analysis was used to determine the candidate lncRNAs with prognostic value. Then, feature selection was carried out by Least absolute shrinkage and selection operator (LASSO) analysis, and seven eligible m6A-related lncRNAs were included to construct the prognostic risk signature. Kaplan–Meier and receiver operating characteristic (ROC) curve analyses were performed to evaluate the predictive capacity of the risk signature both in the training and testing datasets. A nomogram was used to predict 1-year, 2-year, and 3-year overall survival (OS) of AML patients. Next, the expression levels of lncRNAs in the signature were validated in AML samples by qRT-PCR. Functional enrichment analyses were carried out to identify probable biological processes and cellular pathways. The ceRNA network was developed to explore the downstream targets and mechanisms of m6A-related lncRNAs in AML.Results: Seven m6A-related lncRNAs were identified as a prognostic signature. The low-risk group hold significantly prolonged OS. The nomogram showed excellent accuracy of the signature for predicting 1-year, 2-year and 3-year OS (AUC = 0.769, 0.820, and 0.800, respectively). Moreover, the risk scores were significantly correlated with enrichment in cancer hallmark- and malignancy-related pathways and immunotherapy response in AML patients.Conclusion: We developed and validated a novel risk signature with m6A-related lncRNAs which could predict prognosis accurately and reflect the immunotherapy response in AML patients.

Subclones of Bone Marrow CD34+ Cells in Acute Myeloid Leukemia at Diagnosis Confer Responses of Patients to Induction Chemotherapy

BackgroundAcute myeloid leukemia (AML) is a fatal hematopoietic malignancy which has a prognosis that varies with genetic heterogeneity of its hematopoietic stem/progenitor cells(HSCPs). Intensive induction chemotherapy with cytarabine and anthracycline has been the standard of care for newly diagnosed AML, but about 30% of patients have no response to this regimen. The resistance mechanisms require deeper understanding. Methods In our study, using 10× Genomics, a high-throughput single cell RNA sequencing (scRNA-seq) platform, we analyzed the cellular heterogeneity of bone marrow CD34+ cells from newly diagnosed AML patients who were then divided into sensitive and resistant groups according to their responses to induction chemotherapy with cytarabine and anthracycline. ScRNA-seq data for healthy controls were obtained from the GEO database. We verified our findings by the TCGA database, GEO datasets and the multiparameter flow cytometry.ResultsFrom the integrative analysis of 60,402 cells, we established a landscape for single-cell CD34+ cells in AML patients and identified the hematopoietic stem/progenitor cell types based on the lineage signature genes. Interestingly, through recognizing the malignant-like clusters and comparing “resistant group” with “sensitive group”, we found a cell population with specific gene signatures (CRIP1highLGALS1highS100Ashigh) showing features of granulocyte-monocyte progenitors (GMP) was associated with poor prognosis of AML. And two cell populations of AML CD34+ cells marked by CD52+ or CD74+DAP12+ were related to good response of patients to induction therapy, showing characteristics of hematopoietic stem cell (HSC). ConclusionOur study indicates the heterogeneity of AML CD34+ cells confers for outcomes of AML and provides possible biomarkers to predict the response of AML patients to induction chemotherapy.

PHF6 Positively Regulates Transcription of Myeloid Differentiation Genes By Binding at Enhancer Regions

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by uncontrolled division, and differentiation arrest, of hematopoietic stem cells (HSCs) and myeloid progenitors. AML is a genetically heterogeneous disease, with mutations in genes belonging to multiple functional groups. Of these, chromatin regulators and transcriptional factors (TFs) are an important functional group to study because of a lack of targeted AML therapies against these factors. PHD Finger Protein 6 (PHF6) is one such chromatin-associated protein with a yet-unknown molecular mechanism of action. It is mutated in 3-5% of MDS, CMML, and AMLs and 20% of T- ALLs, and is considered a leukemia suppressor. To assess the effects of PHF6 on the AML transcriptome, we generated CRISPR-mediated knockout (KO) clones in THP-1 monocytic AML cell line, and integrated a Dox-inducible PHF6 construct into a PHF6 KO clone, enabling us to conditionally rescue PHF6 expression to parental levels (Fig A & B). RNA-Seq analysis of these knockout and rescue systems revealed that PHF6 expression upregulates genes related to myeloid differentiation and downregulates genes related to hematopoietic stem cells and cell division in myeloid cells. Additionally, loss of PHF6 increased THP-1 proliferation, and restoring PHF6 expression decreased proliferation. We also performed ChIP-Seq for PHF6 in the THP-1 cells and found that PHF6 binds to open and active enhancer regions. Unbiased motif analysis showed that PHF6-bound enhancers (compared to all enhancers genome-wide) were enriched for RUNX1, PU.1, and IRF8 motifs. Metagene plotting of PHF6 ChIP-Seq signal against ChIP-Seq for these TFs showed striking concordance in their binding patterns, indicating that PHF6 co-occupies chromatin with key hematopoietic transcription factors. (Fig C). PHF6 has two extended PHD (ePHD) domains with a similar structure to canonical PHD domains, but with unknown functions. Based on leukemia genome sequencing results from COSMIC, we observed that while nonsense and frameshift mutations of PHF6 (accounting for 2/3 rd of PHF6 mutations, and expected to produce no protein) are distributed throughout the gene body, missense mutations (accounting for 1/3 rd of PHF6 mutations and expected to produce a full-length protein with single amino acid substitution), are concentrated in the second ePHD domain (ePHD2). To assess the functional consequence of mutations in the ePHD2 domain, we generated from a PHF6 KO clone a clone expressing Dox-inducible PHF6 R274Q, the most common missense mutation of PHF6 seen in leukemia. RNA-Seq showed that PHF6 R274Q induction has no downstream effects on the cellular transcriptome, in striking contrast to the effects of wildtype PHF6 induction (Fig D). Additionally, the expression of PHF6 R274Q has no effect on cell growth. These results indicate that the ePHD2-domain mutant PHF6 R274Q is functionally dead (fully or partially), and the ePHD2 domain is critical for PHF6 function. Our results support an important transcriptional role of PHF6 in the myeloid system, involving co-occupancy with TFs at enhancers to promote the transcription of myeloid differentiation genes. This loss of this transcriptional regulation, either through complete PHF6 protein loss or point mutation of its ePHD2 domain, dysregulates myeloid differentiation and contributes to leukemogenesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

ACSL1 Inhibits ALV-J Replication by IFN-Ⅰ Signaling and PI3K/Akt Pathway

J subgroup avian leukosis virus (ALV-J) infection causes serious immunosuppression problems, leading to hematopoietic malignancy tumors in chicken. It has been demonstrated that interferon-stimulated genes (ISGs) could limit ALV-J replication; nevertheless, the underlying mechanisms remain obscure. Here, we demonstrate that Long-chain Acyl-CoA synthetase 1 (ACSL1) is an interferon (IFN)-stimulated gene that specifically restricts the replication of ALV-J due to the higher IFN-I production. More importantly, ACSL1 induces primary monocyte-derived macrophages (MDMs) to pro-inflammatory phenotypic states during ALV-J infection, and ACSL1 mediates apoptosis through the PI3K/Akt signaling pathway in ALV-J-infected primary monocyte-derived macrophages (MDMs). Overall, these results provide evidence that ACSL1 contributes to the antiviral response against ALV-J.

Amyloid deposition in oral cavity and larynx, a single institute experience

Introduction/Objective Amyloidosis is characterized by extracellular accumulation of insoluble amyloid fibril. Amyloid deposition in the head and neck area is rare. Methods/Case Report In this study, we reviewed 34 specimens from 26 patients including: 18 specimens from the larynx and/or pharynx (13 patients) and 16 specimens from the oral cavity (13 patients). The clinical presentation, related laboratory results, and pathologic finding were reviewed. Results (if a Case Study enter NA) Within the 18 laryngeal specimens were: 10 glottic, 4 supraglottic, 3 nasopharyngeal or pharyngeal wall, and 1 subglottic. Of the 16 cases from oral cavity there were 9 lingual, 3 labial, 2 palatine, 1 tonsillar, and 1 alveolar ridge. Ten out of 13 patients with laryngeal amyloid deposition had protein electrophoresis performed and only 3 of the patients had monoclonal light chain detected. Among these three patients, one had multiple myeloma, one had lymphoplasmacytic lymphoma and one had the diagnosis of plasma cell dyscrasia. Interestingly, in the patients with oral cavity amyloidosis, 10 out of 11 patients tested had abnormal findings. Six of the patients had monoclonal light chain, two demonstrated monoclonal peak of IgG kappa, one with IgG lambda and one with IgA lambda. Among these 10 patients, 6 of them had biopsy-proved or history of multiple myeloma, one patient had marginal zone lymphoma, two patients had systematic amyloidosis. Only one patient did not have any malignancy or systematic involvement identified. Conclusion In our small cohort, the most common location of amyloid deposition in the larynx is glottis. When it involves the oral cavity, tongue is the most common location. Compared to the larynx, amyloid deposition in the oral cavity tends to be associated with hematopoietic malignancy or systematic involvement, although this finding needs to be confirmed by a larger scale of study.