Agnostic Administration of Targeted Anticancer Drugs: Looking for a Balance between Hype and Caution.

Many tumors have well-defined vulnerabilities, thus potentially allowing highly specific and effective treatment. There is a spectrum of actionable genetic alterations which are shared across various tumor types and, therefore, can be targeted by a given drug irrespective of tumor histology. Several agnostic drug-target matches have already been approved for clinical use, e.g., immune therapy for tumors with microsatellite instability (MSI) and/or high tumor mutation burden (TMB), NTRK1-3 and RET inhibitors for cancers carrying rearrangements in these kinases, and dabrafenib plus trametinib for BRAF V600E mutated malignancies. Multiple lines of evidence suggest that this histology-independent approach is also reasonable for tumors carrying ALK and ROS1 translocations, biallelic BRCA1/2 inactivation and/or homologous recombination deficiency (HRD), strong HER2 amplification/overexpression coupled with the absence of other MAPK pathway-activating mutations, etc. On the other hand, some well-known targets are not agnostic: for example, PD-L1 expression is predictive for the efficacy of PD-L1/PD1 inhibitors only in some but not all cancer types. Unfortunately, the individual probability of finding a druggable target in a given tumor is relatively low, even with the use of comprehensive next-generation sequencing (NGS) assays. Nevertheless, the rapidly growing utilization of NGS will significantly increase the number of patients with highly unusual or exceptionally rare tumor-target combinations. Clinical trials may provide only a framework for treatment attitudes, while the decisions for individual patients usually require case-by-case consideration of the probability of deriving benefit from agnostic versus standard therapy, drug availability, associated costs, and other circumstances. The existing format of data dissemination may not be optimal for agnostic cancer medicine, as conventional scientific journals are understandably biased towards the publication of positive findings and usually discourage the submission of case reports. Despite all the limitations and concerns, histology-independent drug-target matching is certainly feasible and, therefore, will be increasingly utilized in the future.

Targeted therapeutic options in early and metastatic NSCLC-overview.

The complex therapeutic strategy of non-small cell lung cancer (NSCLC) has changed significantly in recent years. Disease-free survival increased significantly with immunotherapy and chemotherapy registered in perioperative treatments, as well as adjuvant registered immunotherapy and targeted therapy (osimertinib) in case of EGFR mutation. In oncogenic-addictive metastatic NSCLC, primarily in adenocarcinoma, the range of targeted therapies is expanding, with which the expected overall survival increases significantly, measured in years. By 2021, the FDA and EMA have approved targeted agents to inhibit EGFR activating mutations, T790 M resistance mutation, BRAF V600E mutation, ALK, ROS1, NTRK and RET fusion. In 2022, the range of authorized target therapies was expanded. With therapies that inhibit KRASG12C, EGFR exon 20, HER2 and MET. Until now, there was no registered targeted therapy for the KRAS mutations, which affect 30% of adenocarcinomas. Thus, the greatest expectation surrounded the inhibition of the KRAS G12C mutation, which occurs in ∼15% of NSCLC, mainly in smokers and is characterized by a poor prognosis. Sotorasib and adagrasib are approved as second-line agents after at least one prior course of chemotherapy and/or immunotherapy. Adagrasib in first-line combination with pembrolizumab immunotherapy proved more beneficial, especially in patients with high expression of PD-L1. In EGFR exon 20 insertion mutation of lung adenocarcinoma, amivantanab was registered for progression after platinum-based chemotherapy. Lung adenocarcinoma carries an EGFR exon 20, HER2 insertion mutation in 2%, for which the first targeted therapy is trastuzumab deruxtecan, in patients already treated with platinum-based chemotherapy. Two orally administered selective c-MET inhibitors, capmatinib and tepotinib, were also approved after chemotherapy in adenocarcinoma carrying MET exon 14 skipping mutations of about 3%. Incorporating reflex testing with next-generation sequencing (NGS) expands personalized therapies by identifying guideline-recommended molecular alterations.

[Primary cardiac synovial sarcoma: a clinicopathological analysis of five cases].

Objective: To assess the clinicopathological features, immunophenotype, molecular characteristics and differential diagnosis of primary cardiac synovial sarcoma (PCSS). Methods: Five cases of PCSS were collected at Guangdong Provincial People's Hospital from 2008 to 2023, and their clinicopathological features were summarized. Immunohistochemical staining, fluorescence in-situ hybridization (FISH) and next-generation sequencing (NGS) were performed, and relevant literatures were reviewed. Results: The cases were found in four males and one female, ranging in ages from 16 to 51 years (median 30 years). Two cases were located in the pericardium, two in the right ventricle, and one in the left ventricle. Follow-up data were available in four cases. All the four patients died of disease at 3, 7, 13 and 26 months, respectively, after diagnosis. The tumor maximum diameter ranged from 6.0 to 14.0 cm in (mean 10.0 cm). Microscopically, three cases were monophasic and two cases were biphasic. Immunohistochemically, all cases were immunoreactive for EMA, vimentin, bcl-2 and CD56. The tumor cells were variably positive for pan-cytokeratin, SS18-SSX, SOX2, TLE1, CD99, synaptophysin, calretinin and calponin. FISH showed the presence of SS18 rearrangement in all the cases. NGS detected SS18-SSX gene fusion in three cases (SS18-SSX1 in one and SS18-SSX2 in two). Conclusions: PCSS is an exceedingly rare neoplasm, and should be distinguished from other various malignant epithelial and mesenchymal tumors. The clinical history, histopathological and immunohistochemical features, and molecular findings are all essential to the definitive diagnosis of PCSS.

miR-125b reverses cisplatin resistance by regulating autophagy via targeting RORA/BNIP3L axis in lung adenocarcinoma.

The platinum-based chemotherapy is one of the most frequently used treatment protocols for lung adenocarcinoma (LUAD), and chemoresistance, however, usually results in treatment failure and limits its application in the clinic. It has been shown that microRNAs (miRNAs) play a significant role in tumor chemoresistance. In this study, miR-125b was identified as a specific cisplatin (DDP)-resistant gene in LUAD, as indicated by the bioinformatics analysis and the real-time quantitative PCR assay. The decreased serum level of miR-125b in LUAD patients was correlated with the poor treatment response rate and short survival time. MiR-125b decreased the A549/DDP proliferation, and the multiple drug resistance- and autophagy-related protein expression levels, which were all reversed by the inhibition of miR-125b. In addition, xenografts of human tumors in nude mice were suppressed by miR-125b, demonstrating that through autophagy regulation, miR-125b could reverse the DDP resistance in LUAD cells, both in vitro and in vivo. Further mechanistic studies indicated that miR-125b directly repressed the expression levels of RORA and its downstream BNIP3L, which in turn inhibited autophagy and reversed chemoresistance. Based on these findings, miR-125b in combination with DDP might be an effective treatment option to overcome DDP resistance in LUAD.

Vitamin C enhances co-localization of novel TET1 nuclear bodies with both Cajal and PML bodies in colorectal cancer cells.

Deregulation of ten-eleven Translocation protein 1 (TET1) is commonly reported to induce imbalances in gene expression and subsequently to colorectal cancer development (CRC). On the other hand, vitamin C (VitC) improves the prognosis of colorectal cancer by reprogramming the cancer epigenome and limiting chemotherapeutic drug resistance events. In this study, we aimed to characterize TET1-specific subcellular compartments and evaluate the effect of VitC on TET1 compartmentalization in colonic tumour cells. We demonstrated that TET1 is concentrated in coarse nuclear bodies (NB) and 5-hydroxymethylcytosine (5hmC) in foci in colorectal cancer cells (HCT116, Caco-2, and HT-29). To our knowledge, this is the first report of a novel intracellular localization profile of TET1 and its demethylation marker, 5hmC, in CRC cells. Interestingly, we found that TET1-NBs frequently interacted with Cajal bodies, but not with promyelocytic leukaemia (PML) bodies. In addition, we report that VitC treatment of HCT116 cells induces 5hmC foci biogenesis and triggers 5hmC marks to form active complexes with nuclear body components, including both Cajal and PML proteins. Our data highlight novel NB-concentrating TET1 in CRC cells and demonstrate that VitC modulates TET1-NBs' interactions with other nuclear structures. These findings reveal novel TET1-dependent cellular functions and potentially provide new insights for CRC management.

Discovery of Novel Macrocyclic MERTK/AXL Dual Inhibitors.

MERTK and AXL are members of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases that are aberrantly expressed and have been implicated as therapeutic targets in a wide variety of human tumors. Dual MERTK and AXL inhibition could provide antitumor action mediated by both direct tumor cell killing and modulation of the innate immune response in some tumors such as nonsmall cell lung cancer. We utilized our knowledge of MERTK inhibitors and a structure-based drug design approach to discover a novel class of macrocyclic dual MERTK/AXL inhibitors. The lead compound 43 had low-nanomolar activity against both MERTK and AXL and good selectivity over TYRO3 and FLT3. Its target engagement and selectivity were also confirmed by NanoBRET and cell-based MERTK and AXL phosphorylation assays. Compound 43 had excellent pharmacokinetic properties (large AUC and long half-life) and mediated antitumor activity against lung cancer cell lines, indicating its potential as a therapeutic agent.

Synthesis and biological evaluation of 4-imidazolidinone-containing compounds as potent inhibitors of the MDM2/p53 interaction.

Inhibition of MDM2/p53 interaction with small-molecule inhibitors stabilizes p53 from MDM2 mediated degradation, which is a promising strategy for the treatment of cancer. In this report, a novel series of 4-imidazolidinone-containing compounds have been synthesized and tested in MDM2/p53 and MDM4/p53 FP binding assays. Upon SAR studies, compounds 2 (TB114) and 22 were identified as the most potent inhibitors of MDM2/p53 but not MDM4/p53 interactions. Both 2 and 22 exhibited strong antiproliferative activities in HCT-116 and MOLM-13 cell lines harboring wild type p53. Mechanistic studies show that 2 and 22 dose-dependently activated p53 and its target genes and induced apoptosis in cells based on the Western blot, qPCR, and flow cytometry assays. In addition, the antiproliferative activities of 2 and 22 were dependent on wild type p53, while they were not toxic to HEK-293 kidney cells. Furthermore, the on-target activities of 2 were general and applicable to other cancer cell lines with wild type p53. These attributes make 2 a good candidate for future optimization to discover a potential treatment of wild-type p53 cancer.

Study of the effect of sFRP1 protein on molecules involved in the regulation of DNA methylation in CML cell line.

Wnt-signaling pathway plays a crucial role in the pathogenesis and progression of Chronic Myeloid Leukemia (CML). sFRP1 is involved in the suppression of the Wnt-signaling pathway and has been shown to be epigenetically silenced by promoter hypermethylation during CML progression. DNMT3A plays a crucial role in promoter hypermethylation and is responsible for establishing methylation patterns. We aimed to analyze the relationship between sFRP1 expression and DNMT3A, TET1, TET2 and TET3 proteins that are responsible for maintaining cellular methylation patterns; along with miRNAs miR144-3p and miR-767-5p that are known to be associated with these proteins. CML cell lines K562 and K562S which stably expresses sFRP1, were used to compare the changes in miR144-3p and miR-767-5p expression. DNMT3A, TET1, TET2 and TET3 protein levels were analyzed by Western blot. In K562S cells the expression of miR-144-3p and miR-767-5p were decreased along with DNMT3A and TET1 protein levels. On the contrary, TET2 protein was increased. Our results support other reports involving sFRP1 and methylation dynamics; as well as opening new avenues of exploration. Our data supports the conclusion that re-expression of sFRP1 protein alters the expression of factors that play important roles in the overall methylation patterns in the leukemic cell line K562.

BCOR::CREBBP fusion in malignant neuroepithelial tumor of CNS expands the spectrum of methylation class CNS tumor with BCOR/BCOR(L1)-fusion.

Methylation class "CNS tumor with BCOR/BCOR(L1)-fusion" was recently defined based on methylation profiling and tSNE analysis of a series of 21 neuroepithelial tumors with predominant presence of a BCOR fusion and/or characteristic CNV breakpoints at chromosome 22q12.31 and chromosome Xp11.4. Clear diagnostic criteria are still missing for this tumor type, specially that BCOR/BCOR(L1)-fusion is not a consistent finding in these tumors despite being frequent and that none of the Heidelberger classifier versions is able to clearly identify these cases, in particular tumors with alternative fusions other than those involving BCOR, BCORL1, EP300 and CREBBP. In this study, we introduce a BCOR::CREBBP fusion in an adult patient with a right temporomediobasal tumor, for the first time in association with methylation class "CNS tumor with BCOR/BCOR(L1)-fusion" in addition to 35 cases of CNS neuroepithelial tumors with molecular and histopathological characteristics compatible with "CNS tumor with BCOR/BCOR(L1)-fusion" based on a comprehensive literature review and data mining in the repository of 23 published studies on neuroepithelial brain Tumors including 7207 samples of 6761 patients. Based on our index case and the 35 cases found in the literature, we suggest the archetypical histological and molecular features of "CNS tumor with BCOR/BCOR(L1)-fusion". We also present four adult diffuse glioma cases including GBM, IDH-Wildtype and Astrocytoma, IDH-Mutant with CREBBP fusions and describe the necessity of complementary molecular analysis in "CNS tumor with BCOR/BCOR(L1)-alterations for securing a final diagnosis.

Reclassification of a spindle cell sarcoma after identification of a TFG-ROS1 fusion: A case demonstrating the clinical benefit of next-generation sequencing in sarcoma.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal soft tissue sarcomas that often present diagnostic challenges due to their wide and varied morphology. A subset of IMTs have fusions involving ALK or ROS1. The role of next-generation sequencing (NGS) for classification of unselected sarcomas remains controversial. METHODS AND RESULTS: We report a case of a metastatic sarcoma in a 34-year-old female originally diagnosed as an unclassified spindle cell sarcoma with myofibroblastic differentiation and later reclassified as IMT after NGS revealed a TFG-ROS1 rearrangement. Histologically, the neoplasm had spindle cell morphology with a lobulated to focally infiltrative growth pattern with scant inflammatory cell infiltrate. Immunohistochemistry demonstrated focal desmin and variable smooth muscle actin staining but was negative for SOX10, S100, and CD34. Fluorescence in situ hybridization was negative for USP6 or ALK gene rearrangements. NGS revealed a TFG-ROS1 rearrangement and the patient was treated with crizotinib with clinical benefit. CONCLUSIONS: We discuss the role of NGS as well as its potential benefit in patients with unresectable, ALK-negative metastatic disease. Considering this case and previous literature, we support the use of NGS for patients requiring systemic treatment.

Current treatment and novel insights regarding ROS1-targeted therapy in malignant tumors.

BACKGROUND: The proto-oncogene ROS1 encodes an intrinsic type I membrane protein of the tyrosine kinase/insulin receptor family. ROS1 facilitates the progression of various malignancies via self-mutations or rearrangements. Studies on ROS1-directed tyrosine kinase inhibitors have been conducted, and some have been approved by the FDA for clinical use. However, the adverse effects and mechanisms of resistance associated with ROS1 inhibitors remain unknown. In addition, next-generation ROS1 inhibitors, which have the advantage of treating central nervous system metastases and alleviating endogenous drug resistance, are still in the clinical trial stage. METHOD: In this study, we searched relevant articles reporting the mechanism and clinical application of ROS1 in recent years; systematically reviewed the biological mechanisms, diagnostic methods, and research progress on ROS1 inhibitors; and provided perspectives for the future of ROS1-targeted therapy. RESULTS: ROS1 is most expressed in malignant tumours. Only a few ROS1 kinase inhibitors are currently approved for use in NSCLC, the efficacy of other TKIs for NSCLC and other malignancies has not been ascertained. There is no effective standard treatment for adverse events or resistance to ROS1-targeted therapy. Next-generation TKIs appear capable of overcoming resistance and delaying central nervous system metastasis, but with a greater incidence of adverse effects. CONCLUSIONS: Further research on next-generation TKIs regarding the localization of ROS1 and its fusion partners, binding sites for targeted drugs, and coadministration with other drugs is required. The correlation between TKIs and chemotherapy or immunotherapy in clinical practice requires further study.

Chk2 Modulates Bmi1-Deficiency-Induced Renal Aging and Fibrosis via Oxidative Stress, DNA Damage, and p53/TGFß1-Induced Epithelial-Mesenchymal Transition.

Renal aging may lead to fibrosis and dysfunction, yet underlying mechanisms remain unclear. We explored whether deficiency of the Polycomb protein Bmi1 causes renal aging via DNA damage response (DDR) activation, inducing renal tubular epithelial cell (RTEC) senescence and epithelial-mesenchymal transition (EMT). Bmi1 knockout mice exhibited oxidative stress, DDR activation, RTEC senescence, senescence-associated secretory phenotype (SASP), and age-related fibrosis in kidneys. Bmi1 deficiency impaired renal structure and function, increasing serum creatinine/urea, reducing creatinine clearance, and decreasing cortical thickness and glomerular number. However, knockout of the serine-threonine kinase Chk2 alleviated these aging phenotypes. Transcriptomics identified transforming growth factor beta 1 (TGFß1) upregulation in Bmi1-deficient RTECs, but TGFß1 was downregulated upon Chk2 knockout. The tumor suppressor protein p53 transcriptionally activated TGFß1, promoting EMT in RTECs. Bmi1 knockout or oxidative stress (induced with H2O2) increased TGFß1 expression, and EMT in RTECs and was partly reversed by p53 inhibition. Together, Bmi1 deficiency causes oxidative stress and DDR-mediated RTEC senescence/SASP, thus activating p53 and TGFß1 to induce EMT and age-related fibrosis. However, blocking DDR (via Chk2 knockout) or p53 ameliorates these changes. Our study reveals mechanisms whereby Bmi1 preserves renal structure and function during aging by suppressing DDR and p53/TGFß1-mediated EMT. These pathways represent potential targets for detecting and attenuating age-related renal decline.

Construction and experimental validation of a novel ferroptosis-related gene signature for myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by morphological abnormalities and peripheral blood cytopenias, carrying a risk of progression to acute myeloid leukemia. Although ferroptosis is a promising target for MDS treatment, the specific roles of ferroptosis-related genes (FRGs) in MDS diagnosis have not been elucidated. METHODS: MDS-related microarray data were obtained from the Gene Expression Omnibus database. A comprehensive analysis of FRG expression levels in patients with MDS and controls was conducted, followed by the use of multiple machine learning methods to establish prediction models. The predictive ability of the optimal model was evaluated using nomogram analysis and an external data set. Functional analysis was applied to explore the underlying mechanisms. The mRNA levels of the model genes were verified in MDS clinical samples by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The extreme gradient boosting model demonstrated the best performance, leading to the identification of a panel of six signature genes: SREBF1, PTPN6, PARP9, MAP3K11, MDM4, and EZH2. Receiver operating characteristic curves indicated that the model exhibited high accuracy in predicting MDS diagnosis, with area under the curve values of 0.989 and 0.962 for the training and validation cohorts, respectively. Functional analysis revealed significant associations between these genes and the infiltrating immune cells. The expression levels of these genes were successfully verified in MDS clinical samples. CONCLUSION: Our study is the first to identify a novel model using FRGs to predict the risk of developing MDS. FRGs may be implicated in MDS pathogenesis through immune-related pathways. These findings highlight the intricate correlation between ferroptosis and MDS, offering insights that may aid in identifying potential therapeutic targets for this debilitating disorder.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis.

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.

NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization.

BACKGROUND: NOP2/Sun domain 2 (NSUN2) is one of the important RNA methyltransferases catalyzing 5-methylcytosine (m5C) formation and participates in many critical bioprocesses. However, the roles and underlying molecular mechanisms of NSUN2-mediated m5C modification in colorectal cancer (CRC) remain unclear. METHODS: To explore the NSUN2 expression in CRC, fresh tissue samples were collected and Nsun2 knockout mouse was constructed. In vitro and in vivo functional assays were conducted to assess the role of NSUN2. RNA array and bisulfite sequencing were used to investigate the potential targets. The mechanisms of NSUN2 function on SKIL were identified by m5C-methylated-RNA immunoprecipitation and RNA stability assays. Additionally, tissue microarray analysis was conducted and patient-derived tumour xenograft mouse (PDX) models were used to define the potential therapeutic targets. RESULTS: NSUN2 was highly expressed in CRC and correlated with poor CRC patient survival. Moreover, silencing NSUN2 suppressed CRC tumourigenesis and progression in Nsun2 knockout mouse models. In vitro and in vivo studies suggested that NSUN2 promoted colorectal cancer cell growth. Mechanistically, SKI-like proto-oncogene (SKIL) is positively regulated by NSUN2, and the NSUN2-SKIL axis is clinically relevant to CRC. NSUN2 induced m5C modification of SKIL and stabilized its mRNA, which was mediated by Y-box binding protein 1 (YBX1). Elevated SKIL levels increased transcriptional coactivator with PDZ-binding motif (TAZ) activation. CONCLUSIONS: Our findings highlight the importance of NSUN2 in the initiation and progression of CRC via m5C-YBX1-dependent stabilization of the SKIL transcript, providing a promising targeted therapeutic strategy for CRC.

Association of PD-L1 expression with driver gene mutations and clinicopathological characteristics in non-small cell lung cancer: A real-world study of 10 441 patients.

BACKGROUND: Programmed death ligand-1 (PD-L1) expression is a well-known predictive biomarker of response to immune checkpoint blockade in non-small cell lung cancer (NSCLC). However, there is limited evidence of the relationship between PD-L1 expression, clinicopathological features, and their association with major driver mutations in NSCLC patients in Latin America. METHODS: This retrospective study included patients from Argentina with advanced NSCLC, and centralized evaluation of PD-L1 expression concurrently with genomic alterations in the driver genes EGFR, ALK, ROS1, BRAF, and/or KRAS G12C in FFPE tissue samples. RESULTS: A total of 10 441 patients with advanced NSCLC were analyzed. Adenocarcinoma was the most frequent histological subtype (71.1%). PD-L1 expression was categorized as PD-L1 negative (45.1%), PD-L1 positive low-expression 1%-49% (32.3%), and PD-L1 positive high-expression ≥50% (22.6%). Notably, current smokers and males were more likely to have tumors with PD-L1 tumor proportion score (TPS) ≥50% and ≥ 80% expression, respectively (p < 0.001 and p = 0.013). Tumors with non-adenocarcinoma histology had a significantly higher median PD-L1 expression (p < 0.001). Additionally, PD-L1 in distant nodes was more likely ≥50% (OR 1.60 [95% CI: 1.14-2.25, p < 0.01]). In the multivariate analysis, EGFR-positive tumors were more commonly associated with PD-L1 low expression (OR 0.62 [95% CI: 0.51-0.75], p < 0.01), while ALK-positive tumors had a significant risk of being PD-L1 positive (OR 1.81 [95% CI: 1.30-2.52], p < 0.01). CONCLUSIONS: PD-L1 expression was associated with well-defined clinicopathological and genomic features. These findings provide a comprehensive view of the expression of PD-L1 in patients with advanced NSCLC in a large Latin American cohort.

The TAM Subfamily of Receptor Tyrosine Kinases: The Early Years.

The TAMs are a subfamily of receptor tyrosine kinases (RTKs) comprised of three members, Tyro3, Axl and Mer. Evidence in support of the existence of this subfamily emerged from a screen for novel RTKs performed in the laboratory of Dr. Greg Lemke in 1991. A PCR-based approach to selectively amplify tyrosine kinase-specific genes yielded 27 different tyrosine kinase genes, of which 13 were novel (the "Tyros"). Of these, Tyro3, 7 and 12 were more closely related to each other than to any other kinases and it was proposed that they constituted a novel subfamily of RTKs. Additional support for this hypothesis required determining the complete sequences for these receptor tyrosine kinases. By the end of 1991, full-length sequences for Tyro7 (Axl) revealed a unique extracellular domain organization that included two immunoglobulin-like domains and two fibronectin type III repeats. In 1994, the complete sequences for Tyro12 (Mer) and Tyro3 were shown to have an extracellular region domain structure similar to that of Axl. In 1995, Gas6 and Pros1 were reported as ligands for Tyro3 and Axl, setting the stage for functional studies. The Lemke lab and its many trainees have since played leading roles in elucidating the physiological relevance of the TAMs.

GFI1B and LSD1 repress myeloid traits during megakaryocyte differentiation.

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.

Low expression of miR-182 caused by DNA hypermethylation accelerates acute lymphocyte leukemia development by targeting PBX3 and BCL2: miR-182 promoter methylation is a predictive marker for hypomethylation agents + BCL2 inhibitor venetoclax.

BACKGROUND: miR-182 promoter hypermethylation frequently occurs in various tumors, including acute myeloid leukemia, and leads to low expression of miR-182. However, whether adult acute lymphocyte leukemia (ALL) cells have high miR-182 promoter methylation has not been determined. METHODS: To assess the methylation status of the miR-182 promoter, methylation and unmethylation-specific PCR analysis, bisulfite-sequencing analysis, and MethylTarget™ assays were performed to measure the frequency of methylation at the miR-182 promoter. Bone marrow cells were isolated from miR-182 knockout (182KO) and 182 wild type (182WT) mice to construct BCR-ABL (P190) and Notch-induced murine B-ALL and T-ALL models, respectively. Primary ALL samples were performed to investigate synergistic effects of the hypomethylation agents (HMAs) and the BCL2 inhibitor venetoclax (Ven) in vitro. RESULTS: miR-182 (miR-182-5P) expression was substantially lower in ALL blasts than in normal controls (NCs) because of DNA hypermethylation at the miR-182 promoter in ALL blasts but not in normal controls (NCs). Knockout of miR-182 (182KO) markedly accelerated ALL development, facilitated the infiltration, and shortened the OS in a BCR-ABL (P190)-induced murine B-ALL model. Furthermore, the 182KO ALL cell population was enriched with more leukemia-initiating cells (CD43+B220+ cells, LICs) and presented higher leukemogenic activity than the 182WT ALL population. Furthermore, depletion of miR-182 reduced the OS in a Notch-induced murine T-ALL model, suggesting that miR-182 knockout accelerates ALL development. Mechanistically, overexpression of miR-182 inhibited proliferation and induced apoptosis by directly targeting PBX3 and BCL2, two well-known oncogenes, that are key targets of miR-182. Most importantly, DAC in combination with Ven had synergistic effects on ALL cells with miR-182 promoter hypermethylation, but not on ALL cells with miR-182 promoter hypomethylation. CONCLUSIONS: Collectively, we identified miR-182 as a tumor suppressor gene in ALL cells and low expression of miR-182 because of hypermethylation facilitates the malignant phenotype of ALL cells. DAC + Ven cotreatment might has been applied in the clinical try for ALL patients with miR-182 promoter hypermethylation. Furthermore, the methylation frequency at the miR-182 promoter should be a potential biomarker for DAC + Ven treatment in ALL patients.