Oncogenic Hits Research Articles

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

Cutaneous lymphoproliferative disorders after COVID-19 vaccination: clinical presentation, histopathology, and outcomes

Individual reports described lymphoproliferative disorders (LPDs) after COVID-19 vaccination; however, the relationship between cases is unexamined. We aim to determine if there are cases of cutaneous LPDs associated with COVID-19 vaccination and their outcomes. We present a review of world literature, vaccine registries, and two unreported cases of LPDs after COVID-19 vaccination. Review of the medical literature, VAERS, and our two cases reveal predominance of Pfizer-BioNTech vaccine, younger patients, and males. All cases resulted in favorable outcomes. Approximately 84% of cases demonstrated CD30+ positivity in their skin biopsies, suggesting that an antigenic trigger may lead to a type IV adaptive immune response, with clonal expansion of CD30+ T-cells and subsequent oncogenic mutational hits eventuating in transient LPDs. LPDs after COVID-19 vaccination appear in the context of the same vaccines (proportionally to their global market shares), share clinical and pathological findings, and have indolent, self-limited character.

HGG-07. OVEREXPRESSION OF A PLAG-FAMILY TRANSCRIPTION FACTOR DURING MURINE BRAIN DEVELOPMENT CAUSES BRAIN TUMOR FORMATION

Abstract Pediatric-type gliomas are the most common type of central nervous system tumors in children. They are highly diverse regarding their molecular entities and clinical associations, and they differ from their adult counterparts. A subset of pediatric-type high grade gliomas, including H3K27M mutated diffuse midline glioma, overexpresses the developmentally regulated transcription factor gene PLAG1. Two novel tumor types displaying amplifications or fusions with the related PLAGL1 and PLAGL2 genes have also recently been described (PMIDs 36437415, 34355256). How PLAG-family dysregulation drives tumor formation is currently unknown, however. We generated a novel mouse model that overexpresses hPLAG1 during brain development using a Cre-LoxP approach. Mice that overexpress hPLAG1 together with loss of trp53 in the Nestin-Cre lineage develop brain tumors by 3 months of age with 100 % penetrance. Intriguingly, tumors develop in different brain regions, with hotspots in the thalamus or inferior hypothalamus, the latter likely emerging from the brainstem. All mouse brains contain regions in the brainstem, thalamus and the prefrontal cortex with diffuse patterns of PLAG1+; KI67+ proliferating single cells. Using bulk-RNA sequencing, we identified several genes involved in embryonic brain development, which are still active in the tumors of adult mice. We hypothesize that continued overexpression of PLAG1 locks cells in an aberrant developmental state, making them susceptible for further oncogenic hits and ultimately leading to pediatric glioma formation. We are currently using single-nucleus RNA-seq to determine the developmental trajectories of single tumor cells to identify putative cells-of-origin and primary developmental pathways. Our work will elucidate the consequences of PLAG1 overexpression in pediatric brain tumors and its contribution to tumorigenesis. In the future, we hope to identify genetic dependencies of PLAG1 to generate novel treatment options.

High MN1 Expression Is Associated with an Lspc-Enriched Phenotype and Glycolysis Representing a New Vulnerability in Acute Myeloid Leukemia

Despite a steady increase in the number of targeted therapies over the past decades, the 5-year overall survival (OS) rate remains poor in acute myeloid leukemia (AML). Currently, high expression of the oncogenic Meningioma 1 (MN1) is associated with poor OS in AMLs with normal karyotype, and we correlated its expression with a more immature cellular composition in AML samples. Hence, we hypothesized that supplementation of AML cells that usually fail to engraft in xenograft models, such as acute promyelocytic leukemia (APL), with MN1 would promote in vivo expansion in mice allowing the study of potential therapeutic vulnerabilities. The in vitro experiments were performed in human AML cell lines, primary AML cells (n=39), and isolated healthy CD34+ cells. MN1-overexpression (OE) was lentivirally performed. Bioenergetic profiles were evaluated by a Seahorse analyzer. Gene expression was measured by RNA-sequencing/qPCR. Protein levels were measured by immunofluorescence microscopy. Viability and surface protein expression was evaluated by flow cytometry. For in vivo studies, eight-weeks old female NSGS mice were transplanted with MN1-OE primary APL blast cells, and mice were followed for 12 weeks. Cells from the mouse organs were isolated, analyzed, and sorted for human APL blasts (CD45+CD117+CD33+) for ex vivo studies. We show that genetic MN1-OE in APL cells resulted in increased cell survival upon cytotoxic therapy, and decreased sensitivity to ATRA-induced terminal differentiation in short and long term cultures (3/8 days). In healthy CD34+ cells, we observed that MN1-OE was able to increase cell proliferation and stemness properties in CMP-sorted (CD34+CD38+CD123+CD45RA-) but not in GMP-sorted cells (CD34+CD38+CD123+CD45RA+), showing in human models the results from Heuser et al., 2011 in murine MN1-model. Co-expression of MN1+PML-RARα in healthy CD34+ cells resulted in superior expansion with retention of the CD34 marker, in comparison with the single hits. At the molecular level, APL cells with MN1high displayed transcriptional programs associated with "HYPOXIA"/"HSC-UP", whereas MN1low levels correlated with "OXPHOS-UP"/"GMP-UP". Despite no evident differences in Seahorse analysis, MN1-OE cells exhibited increased levels of the glycolytic enzyme PDK1. Functionally, MN1-OE cells were more sensitive to glycolytic inhibitors 2,2-dichloroacetophenone (a PDK1 inhibitor) and 2-deoxyglucose, revealing glycolysis as a metabolic vulnerability in MN1high AMLs, which was further validated in primary AML samples. These data are in line with our previous report indicating that more immature AML subtypes typically display a more glycolytic metabolic profile (Erdem et al, 2022). MN1 is an intrinsically disordered protein containing a long polyQ-stretch that is required for transformation (Riedel et al, 2021). We questioned whether the expression of chaperone proteins would be necessary for the appropriate functioning of MN1. We observed that expression of the DNAJB6 isoform 2 (DNAJB6b) correlated with MN1 expression across a large panel of genetic subtypes of AML, including more immature AML subtypes, such as the TP53-mutant AML but also in APL. shRNA-induced knockdown in AML cells of DNAJB6b resulted in cytosolic retention of MN1, associated with reduced cell survival. In vivo, the patient-derived xenograft model showed engraftment of APL samples upon MN1-OE, with the colonization of secondary organs and splenomegaly, which was not observed in the control. Flow cytometry analysis revealed that MN1-engrafted mice displayed increased levels of APL blasts in comparison with control, which had a large proportion of mature myeloid cells (CD33+CD11b+). Ex vivo screen of engrafted cells revealed increased resistance to ATRA-induced differentiation in MN1-OE sorted blasts. We provide further evidence to support the role of MN1 as an oncogenic hit allowing in vivo studies of difficult-to-engraft AML subtypes. Functionally, high MN1 expression resulted in decreased differentiation, retention of stem-like properties, and metabolic shift towards a more glycolytic state. We demonstrated that DNAJB6b controls the cellular localization of MN1, with its downregulation associated with the loss of MN1 supportive functions. Finally, our findings suggest that targeting glycolysis can be leveraged as a therapeutically actionable mechanism for driving cell death in MN1high AMLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 408: Development of a first-in-class notch transcription factor inhibitor CB-103 to overcome resistance to chemotherapy and targeted therapies

Abstract Human cancers possess striking ability to develop resistance to Standard of Care (SoC) designed to target specific hallmarks of cancer. This capability to develop resistance is in part due to cancer cell’s ability to activate alternative pathways and modulate tumor microenvironment to enhance cell survival, evade cell death and immunosurveillance. One such mechanism of developing resistance to SoC is the activation of the NOTCH pathway. NOTCH signaling is a developmental pathway which is known to be activated in several human cancers as a key oncogenic hit. In addition to its role as an oncogenic driver, activation of NOTCH is also known to induce resistance to several anti-cancer therapeutics in breast cancer and colorectal cancer. NOTCH signaling is known to cooperate with parallel oncogenic signals to impede activity of several drugs as a single agent. Previous agents targeting the NOTCH pathway upstream of the transcription complex were halted early in clinical development due to dose-limiting toxicities.Here we report activity of a novel first-in-class clinical stage CSL-NICD inhibitor in a panel of cell lines, xenograft and PDX models of human cancers in combination with several chemo and targeted therapies. Guided by in vitro and in vivo studies pre-clinical studies, CB-103 is currently being investigated in clinical trials as a single agent and in combination with SoC. Preliminary pre-clinical and clinical data shows desirable safety and efficacy profile of CB-103 enabling clinical development in combination with several therapeutic agents. Citation Format: Michele Vigolo, Charlotte Urech, Sebastien Lamy, Samarpan Majumder, Lucio Miele, Rajwinder Lehal. Development of a first-in-class notch transcription factor inhibitor CB-103 to overcome resistance to chemotherapy and targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 408.

Abstract 3737: DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia

Abstract Infants and young children (> 3 yrs.) harboring ETO2-GLIS2 have the worst outcomes amongst all pediatric acute myeloid leukemia (AML) subtypes, with poor responses to induction therapy, high incidence of relapse, and dismal 5-year survival rates. Previous studies demonstrated that the ETO2-GLIS2 fusion leads to imbalance in the expression of ETS-related transcription factor ERG and GATA1 with neo-super enhancer (SE) activities in a single oncogenic hit (Thirant et al. 2017). ERG was found to co-localize with ETO2-GLIS2 fusion at 90% (372/419) of H3K27ac marked SE-regions and was strongly upregulated (9.44-fold). Herein, we aimed to investigate cooperativity of DNA methylation and chromatin marks on ERG overexpression in this AML subtype. We analyzed transcriptomic data from pediatric ETO2-GLIS2 AML patients (n=40) from dbGAP (phs000465.v19. p8) in comparison to normal bone marrow (NBM) (n=71) samples, confirming upregulation (median log FC: 2.4-fold) in ERG expression in the ETO2-GLIS2 subgroup compared to NBM. Next, we performed an unbiased genome-wide methylation array using Infinium MethylationEPIC 850K-arrays (Illumina). Differences in mean methylation (β-value) at individual CpG sites between NBM and AML samples were considered significant at differences greater than ±10%. We observed reduced methylation at 72% (5/7) CpGs at the ERG-promoter (1500bp upstream of transcription start site) in ETO2-GLIS2 subgroup compared to NBM. Overall median methylation across the promoter was reduced by 12% in the AML subgroup. In comparison, 22 differentially methylated CpGs were observed in the body, of which methylation increased at 68% (15/22) of the CpGs, with median methylation similarly increased by 15% in the AML patients. This represents a classic example of methylation-expression relation, having a promoter demethylation and body-hypermethylation, linked to upregulation of the gene-expression. Next, we investigated the overlaps between CpGs and the distribution of DNAse clusters and H3K27ac marks based on ENCODE data on myeloid leukemia cell line K562. We observed frequent overlaps between demethylated CpGs and DNAse clusters, while H3K27ac broad peaks were coincident with both demethylated CpGs at promoter or hypermethylated CpGs at body. Based on our findings, we thus conclude that DNA-methylation both at the ERG promoter and body is tightly linked to the overlapping chromatin marks of the gene. DNA-methylation might aid in opening of adjacent chromatin on ERG to possibly facilitate SE enrichment and over-expression the gene. Future studies will investigate targeted alterations of DNA-methylation on the gene to test the impact on ERG expression and leukemic growth. Citation Format: Samrat Roy Choudhury, Jordan T. Bird, Stephanie Byrum, Jason E. Farrar. DNA-methylation is tightly linked with super-enhancer marks to upregulate ERG in ETO2-GLIS2 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3737.

The Second Oncogenic Hit Determines the Cell Fate of ETV6-RUNX1 Positive Leukemia.

ETV6-RUNX1 is almost exclusively associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequences of ETV6-RUNX1 expression on cell lineage decisions during B-cell leukemogenesis are completely unknown. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but few carriers develop B-ALL as a result of secondary genetic alterations. The understanding of the mechanisms underlying the first transforming steps could greatly advance the development of non-toxic prophylactic interventions. Using genetic lineage tracing, we examined the capacity of ETV6-RUNX1 to instruct a malignant phenotype in the hematopoietic lineage by cell-specific Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 gene locus. Here we show that, while ETV6-RUNX1 has the propensity to trigger both T- and B-lymphoid malignancies, it is the second hit that determines tumor cell identity. To instigate leukemia, both oncogenic hits must place early in the development of hematopoietic/precursor cells, not in already committed B-cells. Depending on the nature of the second hit, the resulting B-ALLs presented distinct entities that were clearly separable based on their gene expression profiles. Our findings give a novel mechanistic insight into the early steps of ETV6-RUNX1+ B-ALL development and might have major implications for the potential development of ETV6-RUNX1+ B-ALL prevention strategies.

RUNX1 Mutations Lead to a Myeloid Differentiation Block in Association with Enhanced Proliferation By Altering the RUNX1 Transcriptional Program

RUNX1 (AML1) is a transcription factor critically involved in normal haematopoiesis. Inactivating RUNX1 mutations have been frequently described in a variety of myeloid neoplasms, including high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Here, we aimed to functionally and molecularly define the actions of a dominant negative mutant.Overexpression of the RUNX1 mutant S291fs300X (RUNX1 mt) in cord blood (CB) CD34+ cells caused a decline in erythroid colony formation (p= 0.01) which was associated with an immature morphology and an increased number of Annexin V positive erythroid cells. The negative phenotype coincided with a strong reduction in GATA-1 and GATA-2 expression in RUNX1mt CD34+ CB cells after 24-36 hours transduction. On the contrary, the CFU-GM colonies showed a greater than 3-fold enhanced replating capacity compared to control. It appeared that the replating potential was predominantly present in the CD34+CD38- and CD14-CD15- cell population.Upon long-term MS5 stromal co-cultures of transduced RUNX1 mt CB CD34+ cells a rather homogenous cell population was obtained after 8-10 weeks that localized underneath the stromal layer. This cell population was phenotypically defined by CD34+/CD38-. The interactions with the stroma appeared to be important to limit proliferation and retain quiescence. Suspension cultures with myeloid growth factors (IL-3, SCF) of RUNX1 mt CB CD34+ cells provided a similar cell population after 10 weeks of culture. These cells were growth factor-dependent and were phenotypically defined by CD34+/CD38+/CD33+/CD45RA+/CD123+, resembling a GMP phenotype which could be propagated for approximately 20 weeks in suspension. Karyotype analyses of this cell population demonstrated no abnormalities while integration site analysis showed multiple different integration sites that differed between individual experiments, suggesting that the myeloid differentiation block was related to the RUNX1 mutation and not to aspecific integration site effects. Comparable results were obtained with adult normal bone marrow CD34+ cells or peripheral blood CD34+ cells transduced with the RUNX1 mt.Morphological analysis after 8-10 weeks of culture showed monoblastic cells with limited macrophage differentiation but without signs of granulocytic differentiation, even in the presence of G-CSF. This might might be due to downregulation of C/EBPα, one of the key targets of RUNX1. Therefore, week 10 RUNX1 mt CB CD34+ cells were transduced with a retroviral C/EBPα-ER overexpression vector. Re-expression of C/EBPα by tamoxifen induction resulted in a reduction in cell proliferation, decline of undifferentiated blasts and an increase in CD15 expression.In our goal to asses RUNX1 mt target binding and gene expression changes at early time points we used induced pluripotent stem cells (iPS cells) with conditional expression of RUNX1 mt. Expression of RUNX1 mt during early differentiation leads to a differentiation block with the maintenance of CD34+ cells. Genome-wide binding analysis of these RUNX1 mt cells revealed RUNX1 occupancy at similar regions as observed in primary AMLs with a RUNX1 mutation. These binding sites were enriched for RUNX and ETS motifs and associated with genes important for differentiation and chromatin regulation. Moreover, expression analysis revealed a severely altered RUNX1 transcription program in the presence of the RUNX1 mt.Together these results reveal that the RUNX1 mutant S291fs300X, as a single oncogenic hit, induces cell proliferation and a myeloid differentiation block by altering the RUNX1 gene program. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2338: Comparison of genomic alterations in lung cancer specimens from high and low radon zones: Search for a genomic fingerprint

Abstract Introduction: Radon-222 (Rn) is a radioactive gas found in rocks and soil. It emits alpha particles that cause dsDNA breaks and increases potential for carcinogenesis. Rn is the 2nd leading cause of lung cancer in the US after smoking, with EPA estimates of >15000 deaths. A significant portion of land in US has high Rn soil levels; ‘action level' for indoor Rn mitigation is >4pci/L. Rhode Island (RI) contains several high Rn areas. We hypothesize that specific cellular and molecular mechanisms play a role in the neoplastic potential of Rn, detectable as signature genetic profiles. Our aim is to identify molecular patterns in patients (pts) from high Rn zones, by evaluating next generation sequencing (NGS) based assays in advanced lung cancer pts. Methods: Using a commercially available NGS assay, we retrospectively analyzed genomic DNA alterations in 324 genes from FFPE specimens of 159 stage IV lung cancer pts treated at Lifespan Cancer Institute at Brown University from 2014- 2019. Based on EPA Rn maps, we identified counties in RI with high indoor Rn levels (>4 pci/L), and compared the gene mutation patterns with those residing in low Rn areas (<4 pci/L). Results: Of the 159 pts (median age= 58), 35 (22%) belonged to high Rn and 124 (78%) to low Rn zones. Both groups had predominantly adenocarcinoma histology and high smoking prevalence (75-80%). Most prevalent genomic alterations in both cohorts were TP53, KRAS and CDKN2A mutations. In the high Rn group, we identified more frequent recurrent mutations in two DNA repair genes: ATM (11% in high Rn vs 1% in low Rn areas, p=0.00086) and CHEK2 (6% vs 0%, respectively, p=0.047). We then classified all detected genomic alterations into major pathways implicated in lung carcinogenesis; DNA repair, cell cycle, tyrosine kinase, and apoptosis. The only observed statistically significant difference in the frequency of mutations was for the DNA repair pathway (29% in high Rn vs 13% in low Rn, p=0.038). Conclusion: To our knowledge, this is the first report of a novel and mechanistically unique insight into the pathobiology of Rn induced lung cancer. Our observations suggest that Rn induced dsDNA breaks may constitute an oncogenic hit in cells which are unable to efficiently repair them because of DNA repair pathway mutations (particularly ATM and CHEK2). We are further testing this “second hit” hypothesis in a larger nationwide dataset comparing NGS profiles of lung cancers from low and high Rn areas in the US. Top 10 mutated genes in High and Low Radon groups (rank; frequency, %)High RnLow RnP value1. TP53 (63)1. TP53 (70)0.422. KRAS (26)2. KRAS (28)0.833. CDKN2A (23)3. CDKN2A (24)0.994. RB1 (17)4. RB1 (20)0.815. EGFR (14)7. EGFR (10)0.546. CDKN2B (14)6. CDKN2B (15)0.997. STK11 (11)5. STK11 (19)0.338. RBM10 (11)-. RBM10 (3)0.07-. SMARCA4 (3)8. SMARCA4 (8)0.469. NF1 (11)9. NF1 (7)0.4910. ATM (11)-. ATM (1)0.008-. MYC (6)10. MYC (5)0.99 Citation Format: Hina Khan, Harish Saiganesh, Adam J. Olszewski, Christopher G. Azzoli, Howard Safran. Comparison of genomic alterations in lung cancer specimens from high and low radon zones: Search for a genomic fingerprint [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2338.

Type 1 Interferon Responses Underlie Tumor-Selective Replication of Oncolytic Measles Virus.

The mechanism of tumor-selective replication of oncolytic measles virus (MV) is poorly understood. Using a stepwise model of cellular transformation, in which oncogenic hits were additively expressed in human bone marrow-derived mesenchymal stromal cells, we show that MV-induced oncolysis increased progressively with transformation. The type 1 interferon (IFN) response to MV infection was significantly reduced and delayed, in accordance with the level of transformation. Consistently, we observed delayed and reduced signal transducer and activator of transcription (STAT1) phosphorylation in the fully transformed cells. Pre-treatment with IFNβ restored resistance to MV-mediated oncolysis. Gene expression profiling to identify the genetic correlates of susceptibility to MV oncolysis revealed a dampened basal level of immune-related genes in the fully transformed cells compared to their normal counterparts. IFN-induced transmembrane protein 1 (IFITM1) was the foremost basally downregulated immune gene. Stable IFITM1 overexpression in MV-susceptible cells resulted in a 50% increase in cell viability and a significant reduction in viral replication at 24h after MV infection. Overall, our data indicate that the basal reduction in functions of the type 1 IFN pathway is amajor contributor to the oncolytic selectivity of MV. In particular, we have identified IFITM1 as a restriction factor for oncolytic MV, acting at early stages of infection.

The acute myeloid leukemia associated AML1-ETO fusion protein alters the transcriptome and cellular progression in a single-oncogene expressing in vitro induced pluripotent stem cell based granulocyte differentiation model.

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect normal hematopoiesis. The analysis of human AMLs has mostly been performed using end-point materials, such as cell lines and patient derived AMLs that also carry additional contributing mutations. The molecular effects of a single oncogenic hit, such as expression of the AML associated oncoprotein AML1-ETO on hematopoietic development and transformation into a (pre-) leukemic state still needs further investigation. Here we describe the development and characterization of an induced pluripotent stem cell (iPSC) system that allows in vitro differentiation towards different mature myeloid cell types such as monocytes and granulocytes. During in vitro differentiation we expressed the AML1-ETO fusion protein and examined the effects of the oncoprotein on differentiation and the underlying alterations in the gene program at 8 different time points. Our analysis revealed that AML1-ETO as a single oncogenic hit in a non-mutated background blocks granulocytic differentiation, deregulates the gene program via altering the acetylome of the differentiating granulocytic cells, and induces t(8;21) AML associated leukemic characteristics. Together, these results reveal that inducible oncogene expression during in vitro differentiation of iPS cells provides a valuable platform for analysis of aberrant regulation in disease.

Synthetic lethality in multiple myeloma harboring double oncogenic hits of 17p13(del) and 1q21(amp)

Recurrent chromosomal aberrations are the main hallmark of multiple myeloma (MM). A recent large scale genome-wide analysis has unveiled that the co-occurrence of 17p13(del) and 1q21(amp) lesions constitute a novel high-risk disease group. Within 17p13, lies TP53 that maintains the genomic integrity by keeping the double stranded DNA damage (DSB) pathway in check. On the other hand, ADAR1 is a critical gene within 1q21 and we have recently reported that its direct RNA editing mechanism on NEIL1 (base-excision repair (BER) gene) caused defective single stranded DNA breaks (SSB) repair, resulting in CHK1 activation.

Hh signaling from de novo organizers drive lgl neoplasia in Drosophila epithelium

The Hedgehog (Hh) morphogen regulates growth and patterning. Since Hh signaling is also implicated in carcinogenesis, it is conceivable that de novo Hh-secreting organizers, if formed in association with oncogenic hit could be tumor-cooperative. Here we validate this hypothesis using the Drosophila model of cooperative epithelial carcinogenesis. We generate somatic clones with simultaneous loss of tumor suppressor, Lgl, and gain of the posterior compartment selector, Engrailed (En), known to induce synthesis of Hh. We show that lgl UAS-en clones in the anterior wing compartment trigger Hh signaling cascade via cross-talk with their Ci-expressing wild type cell neighbors. Hh-Dpp signaling from clone boundaries of such ectopically formed de novo organizers in turn drive lgl carcinogenesis. By contrast, Ci-expressing lgl clones transform by autocrine and/or juxtracine activation of Hh signaling in only the posterior compartment. We further show that sequestration of the Hh ligand or loss of Dpp receptor, Tkv, in these Hh-sending or –receiving lgl clones arrested their carcinogenesis. Our results therefore reveal a hitherto unrecognized mechanism of tumor cooperation by developmental organizers, which are induced fortuitously by oncogenic hits.

Co-expression of MDM2 and CDK4 in transformed human mesenchymal stem cells causes high-grade sarcoma with a dedifferentiated liposarcoma-like morphology

Amplification and overexpression of MDM2 and CDK4 are well-known diagnostic criteria for well-differentiated liposarcoma (WDLPS)/dedifferentiated liposarcoma (DDLPS). Although it was reported that the depletion of MDM2 or CDK4 decreased proliferation in DDLPS cell lines, whether MDM2 and CDK4 induce WDLPS/DDLPS tumorigenesis remains unclear. We examined whether MDM2 and/or CDK4 cause WDLPS/DDLPS, using two types of transformed human bone marrow stem cells (BMSCs), 2H and 5H, with five oncogenic hits (overexpression of hTERT, TP53 degradation, RB inactivation, c-MYC stabilization, and overexpression of HRASv12). In vitro functional experiments revealed that the co-overexpression of MDM2 and CDK4 plays a key role in tumorigenesis by increasing cell growth and migration and inhibiting adipogenic differentiation potency when compared with the sole expression of MDM2 or CDK4. Using mouse xenograft models, we found that the co-overexpression of MDM2 and CDK4 in 5H cells with five additional oncogenic mutations can cause proliferative sarcoma with a DDLPS-like morphology in vivo. Our results suggest that the co-overexpression of MDM2 and CDK4, along with multiple genetic factors, increases the tendency for high-grade sarcoma with a DDLPS-like morphology in transformed human BMSCs by accelerating their growth and migration and blocking their adipogenic potential.

Desperately seeking a home marrow niche for T-cell acute lymphoblastic leukaemia.

T-cell acute leukemia is a hematologic malignancy that results from the progressive acquisition of genomic abnormalities in T-cell progenitors/precursors. T-ALL is commonly thought to originate from the thymus albeit recent literature describes the possible acquisition of the first oncogenic hits in hematopoietic progenitor cells of the bone marrow (BM). The journey of T-ALL from its arising to full blown expansion meets different microenvironments, including the BM in which leukemic cells settle down early after the disease spreading. We take advantage of recent literature to give an overview of important cells and factors that participate in T-ALL, especially in the BM, arguing in favor of a home marrow niche for this rare leukemia.

RNA Editors and DNA Mutators: Cancer Heterogeneity Through Sequence Diversification

Cancer development and progression is strongly associated with somatic mutations. From oncogenic hits that initiate the primary tumor formation till metastasis, the tumor mutational burden (TBM) plays a prominent role in disease progression for the vast majority of cancer types. Heterogeneous mutational loads or genetic heterogeneity not only between individuals, but also between tumor cells, are causal to transcriptomic and proteomic discrepancies and to phenotypic diversity. In addition to mutations, RNA editing enzymes (ADAR and AID/APOBEC family deaminases) catalyze A-to-I and C-to-U base changes within RNA transcripts, increasing informational heterogeneity. Because some of these enzymes have the ability to also mutate DNA, they are considered central drivers of inter-tumor heterogeneity either at the DNA level or at the level of RNA. In this review, we connect genome/transcriptome diversification with tumorigenesis and suggest specific roles for these DNA/RNA deaminases in the context of tumorigenesis both through genome level events (microsatellite instability (MSI), TMB) and through transcriptome level events as these converge into a common sequence diversification strategy.

Modeling of the Epigenome of the Cell-of-Origin Identifies Cancer-Specific DNA Methylation Patterns in CLL

Modeling of the Epigenome of the Cell-of-Origin Identifies Cancer-Specific DNA Methylation Patterns in CLL

Abstract A01: Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer

Abstract Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. For these reasons, new technologies are needed to expand the genetic toolbox of cancer biologists and allow a more rapid and systematic in vivo interrogation of gene perturbations. In this regard, the advent of CRISPR/Cas9 technologies for somatic genome editing has already paved the way for a new generation of non-germline animal tumor models. For example, liver-specific gene disruption was achieved by transient delivery of components of the CRISPR/Cas9 system in the tail veins of mice, leading to hepatocellular carcinoma (Xue et al., 2014; Weber et al., 2015). Similar approaches have been used to deliver targeted oncogenic mutations to the lung (Platt et al., 2014; Sánchez-Rivera et al., 2014), brain (Zuckermann et al., 2015), and pancreas (Chiou et al., 2015). We have previously shown that the mammary tissue is also amenable to somatic gene editing using intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in the mammary glands of female mice carrying conditional predisposing alleles (Annunziato et al., 2016). This approach was successfully applied to validate in vivo candidate tumor suppressors implicated in invasive lobular carcinoma (Kas et al., 2017), but it is conceivable that more breast cancer subtypes can be modeled via somatic engineering in mice with distinct predisposing mutations. In this study, we show how somatic engineering via intraductal injection of lentiviruses can be used to develop innovative mouse models of triple-negative breast cancer (TNBC), one of the subtypes with the poorest prognosis in the clinics. Using intraductal injection of Cre-encoding lentiviruses in mice with conditional Brca1 and Trp53 alleles (B1P), we were able to target TNBC-initiating cells from the basal compartment, and induce tumors that retained all the hallmark features of the transgenic Cre-driven tumor counterparts, including latency, histopathology, and DNA copy-number variation (CNV) pattern. Moreover, we could dramatically accelerate tumorigenesis when lentiviral Cre was injected in B1PM mice that harbored, in addition to conditional Brca1 and Trp53 alleles, a conditional knock-in allele overexpressing the Myc oncogene, a candidate driver in BRCA1-associated cancers. Alternatively, somatic Myc expression and loss of BRCA1 and p53 was induced in the mammary glands of B1P mice via intraductal injection of lentiviral vectors encoding Myc and Cre under a viral promoter. In both cases the resulting tumors displayed a reshaped CNV profile that strongly implicated a limited number of recurrent focal amplifications and deletions in the tumorigenic cascade. We reiterated the process by lentiviral overexpression of these candidate driver oncogenes and Cre in the mammary glands of B1PM mice, or by achieving in situ CRISPR/Cas9-mediated somatic gene disruption of one or multiple candidate tumor-suppressor genes in Cas9-transgenic WB1P-Cas9 mice. This versatile somatic platform allows for rapid, flexible, and multiplexable in vivo testing of putative oncogenic hits and deals with the need for increasingly complex mouse models while avoiding the bottlenecks of classical transgenesis. Notably, we show here that we can extend the applicability of non-germline breast cancer modeling in mice to produce genetically and histologically accurate TNBC, which allows in vivo investigation of the underlying biology as well as rapid generation of tailored preclinical models for this aggressive breast cancer subtype. Citation Format: Stefano Annunziato, Julian R. de Ruiter, Chiara S. Brambillasca, Sjors M. Kas, Federica Ferrante, Catrin Lutz, Bjorn Siteur, Bas van Gerwen, Marieke van de Ven, Martine H. van Miltenburg, Linda Henneman, Jos Jonkers. Somatic engineering of the mammary gland for the development of novel mouse models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A01.

Modeling Glioma with Human Embryonic Stem Cell-Derived Neural Lineages.

Gliomas are malignant primary tumors of the central nervous system. Their cell-of-origin is thought to be a neural progenitor or stem cell that acquires mutations leading to oncogenic transformation. Thanks to advances in human stem cell biology, it has become possible to derive human cell types that represent putative cells-of-origin in vitro and model the gliomagenesis process by systematically introducing genetic alterations in these human cells. Here, we present methods to derive human neural stem cells (NSCs) from human embryonic stem cells (hESCs). Because these NSCs are genetically unmodified at baseline, they can be used as a cellular platform to study the effects of individual oncogenic hits in a well-controlled manner in the backdrop of a human genetic background. We also present some key applications of these NSCs, which include their transduction with lentiviral vectors, their neuroglial differentiation and xenografting methods into immunocompromised mice to assess in vivo behavior.

Low-Grade Astrocytoma Mutations in IDH1, P53, and ATRX Cooperate to Block Differentiation of Human Neural Stem Cells via Repression of SOX2

Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness invivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis.