Isogenic Clones Research Articles

Double-Stranded RNA Viruses Are Released From Trichomonas vaginalis Inside Small Extracellular Vesicles and Modulate the Exosomal Cargo.

Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40–70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis.

Investigating the Molecular and Phenotypic Differences in Pancreatic Neuroendocrine Tumor (PanNET) Cells with Functional Loss of Either ATRX or DAXX

Telomeres are hexameric DNA repeats at the ends of linear chromosomes that protect genomic integrity. Telomeres shorten after each cell division until a critically shortened length is reached, whereby the cell enters a state of cellular senescence. Cancer cells must overcome this replicative barrier by either upregulating the enzyme telomerase or by using the Alternative Lengthening of Telomeres (ALT) pathway, which uses homology‐directed repair to maintain telomere lengths. ALT is present in ~10% of cancers, yet the exact underlying mechanisms are still being elucidated. However, two known drivers of ALT are loss of function mutations in either ATRX or DAXX, genes that encode a chromatin remodeling complex. Mutations in these genes were initially identified and linked to ALT in pancreatic neuroendocrine tumors (PanNETs), which are the second most common malignancy of the pancreas. This study evaluated the molecular and phenotypic differences between parental PanNET cells (BON1 and QGP1) and isogenic clones with either ATRX or DAXX knocked out via CRISPR‐Cas9 gene editing. The matched parental and isogenic clones were assessed for cell proliferation (MTS assay), clonogenic survival (colony formation assay), and invasion potential (Matrigel invasion assay). Preliminary results for QGP1 cells indicate that the ATRXKO clones show decreased proliferation and colony formation, as well as alterations in invasion potential compared to the parental cell line, thereby suggesting that functional loss of ATRX alters these phenotypic properties. Future studies will help to contribute to the ongoing efforts to understand the underlying mechanisms of ALT and work towards the overall goal of identifying potential biomarkers for early detection, prognosis, and targeted therapies for ALT‐positive cancers.

Cytidine nucleoside analog is an effective antiviral drug against Trichomonasvirus.

Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology. 7-Deaza-2'-C-methyladenosine (7d2CMA) and 2'-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions. We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10μM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines. 2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection.

A clinically relevant heterozygous ATR mutation sensitizes colorectal cancer cells to replication stress

Colorectal cancer (CRC) ranks third among the most frequent malignancies and represents the second most common cause of cancer-related deaths worldwide. By interfering with the DNA replication process of cancer cells, several chemotherapeutic molecules used in CRC therapy induce replication stress (RS). At the cellular level, this stress is managed by the ATR-CHK1 pathway, which activates the replication checkpoint. In recent years, the therapeutic value of targeting this pathway has been demonstrated. Moreover, MSI + (microsatellite instability) tumors frequently harbor a nonsense, heterozygous mutation in the ATR gene. Using isogenic HCT116 clones, we showed that this mutation of ATR sensitizes the cells to several drugs, including SN-38 (topoisomerase I inhibitor) and VE-822 (ATR inhibitor) and exacerbates their synergistic effects. We showed that this mutation bottlenecks the replication checkpoint leading to extensive DNA damage. The combination of VE-822 and SN-38 induces an exhaustion of RPA and a subsequent replication catastrophe. Surviving cells complete replication and accumulate in G2 in a DNA-PK-dependent manner, protecting them from cell death. Together, our results suggest that RPA and DNA-PK represent promising therapeutic targets to optimize the inhibition of the ATR-CHK1 pathway in oncology. Ultimately, ATR frameshift mutations found in patients may also represent important prognostic factors.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

IPSC reprogramming-mediated aneuploidy correction in autosomal trisomy syndromes.

Trisomy 21, 18, and 13 are the major autosomal aneuploidy disorders in humans. They are mostly derived from chromosome non-disjunction in maternal meiosis, and the extra trisomic chromosome can cause several congenital malformations. Various genes on the trisomic chromosomes are intricately involved in the development of disease, and fundamental treatments have not yet been established. However, chromosome therapy has been developed to correct the extra chromosome in cultured patient cells, and it was recently reported that during reprogramming into iPSCs, fibroblasts from a Down syndrome patient lost the extra chromosome 21 due to a phenomenon called trisomy-biased chromosome loss. To gain preliminary insights into the underlying mechanism of trisomy rescue during the early stages of reprogramming, we reprogrammed skin fibroblasts from patients with trisomy syndromes 21, 18, 13, and 9 to iPSC, and evaluated the genomes of the individual iPSC colonies by molecular cytogenetic techniques. We report the spontaneous correction from trisomy to disomy upon cell reprogramming in at least one cell line examined from each of the trisomy syndromes, and three possible combinations of chromosomes were selected in the isogenic trisomy-rescued iPSC clones. Single nucleotide polymorphism analysis showed that the trisomy-rescued clones exhibited either heterodisomy or segmental uniparental isodisomy, ruling out the possibility that two trisomic chromosomes were lost simultaneously and the remaining one was duplicated, suggesting instead that one trisomic chromosome was lost to generate disomic cells. These results demonstrated that trisomy rescue may be a phenomenon with random loss of the extra chromosome and subsequent selection for disomic iPSCs, which is analogous to the karyotype correction in early preimplantation embryos. Our finding is relevant for elucidating the mechanisms of autonomous karyotype correction and future application in basic and clinical research on aneuploidy disorders.

Comparative Analysis of the APOL1 Variants in the Genetic Landscape of Renal Carcinoma Cells.

Simple SummaryRenal cell carcinoma (RCC) occurs at higher frequency in individuals of African ancestry, with well-recorded documentation in this community. This is most prominent in the context of chronic kidney disease. In turn, many forms of progressive chronic kidney disease are more common in populations of Sub-Saharan African ancestry. This disparity has been attributed to well-defined allelic variants and has risen in the parental populations to high frequency under evolutionary pressure. Mechanisms of increased kidney disease risk and cell injury, causally associated with these APOL1 gene variants, have been extensively studied. Most studies have compared the effects of ectopic overexpression of the parental non-risk APOL1 with the mutated risk variants in cellular and organismal platforms. In the current study, we have used CRISPR/Cas9 genetic engineering to knock out or modify the sequence of endogenous APOL1 in RCC to mimic and examine the effects of these naturally occurring kidney disease risk allelic variants. Remarkably, these modifications to endogenous APOL1 genes in RCC resulted in a set of prominent effects on mitochondrial integrity and metabolic pathways and disrupted tumorigenesis. These findings both clarify pathways of cell injury of APOL1 risk variants in cells of kidney origin and motivate further studies to examine the potential central role of APOL1 in the pathogenesis of renal cell carcinoma and its relation to chronic kidney disease in genotypically at-risk African ancestry individuals.Although the relative risk of renal cell carcinoma associated with chronic kidney injury is particularly high among sub-Saharan African ancestry populations, it is unclear yet whether the APOL1 gene risk variants (RV) for kidney disease additionally elevate this risk. APOL1 G1 and G2 RV contribute to increased risk for kidney disease in black populations, although the disease mechanism has still not been fully deciphered. While high expression levels of all three APOL1 allelic variants, G0 (the wild type allele), G1, and G2 are injurious to normal human cells, renal carcinoma cells (RCC) naturally tolerate inherent high expression levels of APOL1. We utilized CRISPR/Cas9 gene editing to generate isogenic RCC clones expressing APOL1 G1 or G2 risk variants on a similar genetic background, thus enabling a reliable comparison between the phenotypes elicited in RCC by each of the APOL1 variants. Here, we demonstrate that knocking in the G1 or G2 APOL1 alleles, or complete elimination of APOL1 expression, has major effects on proliferation capacity, mitochondrial morphology, cell metabolism, autophagy levels, and the tumorigenic potential of RCC cells. The most striking effect of the APOL1 RV effect was demonstrated in vivo by the complete abolishment of tumor growth in immunodeficient mice. Our findings suggest that, in contrast to the WT APOL1 variant, APOL1 RV are toxic for RCC cells and may act to suppress cancer cell growth. We conclude that the inherent expression of non-risk APOL1 G0 is required for RCC tumorigenicity. RCC cancer cells can hardly tolerate increased APOL1 risk variants expression levels as opposed to APOL1 G0.

Premature Termination Codon in 5' Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation.

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

Non-genetic determinants of malignant clonal fitness at single-cell resolution

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of thesecretory leukocyte peptidase inhibitorgene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues andimmune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

TP53 Null AML Line Does Not Selectively Expand Under Lenalidomide Treatment

Lenalidomide is an immunomodulatory therapeutic (IMiD) with known anti-proliferative and anti-angiogenic effects, and is an approved therapy for both 5q- Myelodysplastic Syndromes and Multiple Myeloma (MM). This thalidomide analog is known to retarget the E3 ubiquitin ligase cereblon to differential ubiquitination, and therefore degradation, of specific substrates. While lenalidomide maintenance is known to promote progression-free survival, increased rates of therapy-related myeloid neoplasms (TMNs) have been observed in MM patients on IMiD maintenance therapy following autologous stem cell transplant. Of note, TP53 is the most commonly mutated gene in TMN arising after lenalidomide therapy (Mouhieddine et al. 2020). TP53 is also recurrently mutated in TMNs following chemotherapy, suggesting that genotoxic stress results in the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations in TP53 (TN Wong et al. 2018). Based on these observations, we hypothesize that lenalidomide may contribute to the development of TMN by providing a fitness advantage to HSPCs carrying TP53 mutations.To test this hypothesis, we generated TP53 -/- deficient MOLM13 AML cells using CRISPR-Cas9 gene editing. We confirmed guide editing efficiency by next generation sequencing of a bulk edited pool of cells and generated isogenic clones via outgrowth of singly sorted cells, in an accepted approach for the field (Boettcher et al. 2019). We validated clones via sequencing and western blotting, and identified no differences in basal proliferation characteristics. Next, we further compared TP53 -/- deficient clones' with TP53 +/+ clones' response to cisplatin treatment (0.05-10µM) and observed a 2.8 fold increase in IC50 for TP53 -/- deficient clones. Satisfied that TP53 -/- isogenic clones successfully recapitulated this known resistance phenotype, we examined the response to lenalidomide by mixing TP53 -/- and TP53 +/+(AAVS1 edited control) clones and measuring expansion under lenalidomide treatment. However, we observed substantial inter-clone heterogeneity, with widely varying cellular proliferation characteristics following clone mixing, despite our prior validation. Therefore, we shifted to using bulk edited MOLM-13 cells in order to mitigate the potential impact of off-target effects and strong selective pressure of singly sorted clones. We edited a pool of cells at either TP53 or the safe harbor locus AAVS1 , allowed cells to recover, and mixed the two edited populations before treating with PBS, vehicle (DMSO), 1µm or 10µM lenalidomide for up to 28 days. We sequenced both AAVS1 and TP53 loci to measure cellular expansion by tracking the fraction of unedited, safe harbor edited, and TP53-/- genomic DNA at 0, 3, 7, and 28 days. Contrary to our hypothesis, TP53 -/- cells did not expand over time in response to lenalidomide treatment, and we did not detect a difference in TP53 -/- cell expansion with lenalidomide treatment compared to vehicle control (Figure 1). While we did not detect a cell intrinsic effect of TP53 mutations on lenalidomide response, it is possible that there are cell extrinsic effects, such as alterations in the bone marrow microenvironment, that contribute to the development of TP53-mutant TMN. It is also possible that a more prolonged exposure of lenalidomide is needed to see a significant effect of TP53-mutant cell expansion. This is relevant, since patients may be maintained on lenalidomide therapy for years. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ATR/CHK1/WEE1 Dependency in SRSF2-Mutated MDS/AML

Background: Mutations in splicing factor gene SRSF2 are recurrent drivers in 5-15 % of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A key property of SRSF2 mutations is that they occur early in the pathogenesis of MDS and are therefore present in all tumor cells in a patient. This property makes targetable vulnerabilities caused by SRSF2 mutations exceptionally important as they provide a way to inhibit the whole tumor. We previously demonstrated that splicing factor mutations induce R-loop-dependent activation of ATR, rendering cells sensitive to ATR inhibition. R-loops are transcription intermediates consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. Accumulation of aberrant R-loops induces the ATR kinase, which activates the G2-M cell cycle checkpoint via CHK1- and WEE1-mediated signaling. In normal cells, activation of the G2-M checkpoint halts the cell cycle until R-loops have been resolved. When the ATR pathway is inhibited, checkpoint activation does not occur, causing cells with unresolved R-loops to proceed to mitosis, resulting in DNA damage and cell death. We, therefore, sought to assess primary human MDS/AML samples for sensitivity to perturbation of the ATR/CHK1/WEE1 pathway and identify mechanisms of resistance.Methods: Sensitivity of 147 AML patient samples to 515 oncology drugs was tested ex vivo. Bone marrow mononuclear cells were incubated with 5 concentrations of each drug for 72 h followed by measurement of cell viability by CTG assay. Somatic mutations were identified by exome sequencing of matched leukemic bone marrow and skin biopsy samples.Isogenic K562 cell line clones carrying SRSF2 P95H/L/R mutations were generated using CRISPR/Cas9 editing. The presence of SRSF2 mutation was confirmed by CRISPR-sequencing and expression by whole transcriptome RNA-sequencing. Drug sensitivity of the K562 clones with and without SRSF2 mutation was determined by incubating cells with 16 concentrations of prexasertib, SRA-737, adavosertib, or BAY-1895344, followed by determination of cell viability by the MTS assay.Results: Analysis of ex vivo drug sensitivities in AML patient samples identified vulnerability to CHK1 and WEE1 inhibition in SRSF2-mutated AML: SRSF2 mutation is associated with sensitivity to the CHK1 inhibitors prexasertib (p = 0.006) (Fig 1 A and B) and PF-00477736 (p = 0.002) and the WEE1 inhibitor adavosertib (p = 0.003).To establish whether the isogenic SRSF2-mutant K562 cell line models recapitulate known downstream aberrations associated with SRSF2 mutations in patients, we analyzed gene expression and splicing. SRSF2 contains an RNA binding domain with affinity to CCNG or GGNG exonic splicing enhancer sequences. Similar to what has been observed in patients, the K562 clones with SRSF2 mutation show reduced use of GGNG sequence motifs at skipped exons. These results demonstrate that isogenic K562 clones recapitulate known alterations caused by mutant SRSF2.To determine whether SRSF2 mutations induce sensitivity to inhibition of ATR, CHK1, and WEE1, we tested 10 isogenic SRSF2 mutant and 4 wild-type K562 clones. Cells with SRSF2 mutation show increased sensitivity to ATR/CHK1/WEE1 inhibition (Fig 1C). We found no significant difference in drug sensitivity between clones carrying SRSF2 P95H/L/R substitutions. Clones with higher SRSF2 mutant allele dosage are more sensitive (Fig 1D).We identified a subset of SRSF2 mutated AML samples that were resistant to CHK1 and WEE1 inhibition. All resistant AML have co-occurring RUNX1 mutations (Fig 1B). In AML, RUNX1 mutations are associated with therapy resistance, suggesting that these mutations contribute to drug resistance. To test whether RUNX1 mutations induce resistance to ATR/CHK1/WEE1 inhibition in SRSF2-mutant leukemia, we introduced RUNX1 loss-of-function mutations in isogenic K562 carrying SRSF2 mutations. Candidate resistance factors identified by ATAC and RNA-sequencing will be validated in functional assays.Conclusions: Our results indicate that SRSF2-mutated leukemia harbor a vulnerability to the inhibition of ATR, CHK1, and WEE1 kinases. Cell line models indicate that sensitivity is similar across mutant alleles and dependent on allelic copy number. Several ATR/CHK1 and WEE1 inhibitors are in development, and our results suggest that these compounds could be effective treatments for SRSF2-mutated MDS and AML. [Display omitted] DisclosuresGraubert: astrazeneca: Research Funding; Janssen: Research Funding; Calico: Research Funding.

Radiosensitization of calreticulin-overexpressing human glioma cell line by the polyphenolic acetate 7, 8-diacetoxy-4-methylcoumarin.

Calreticulin (CRT), an endoplasmic reticulum-resident protein generally overexpressed in cancer cells, is associated with radiation resistance. CRT shows higher transacetylase activity, as shown by us earlier, in the presence of the polyphenolic acetates (like 7, 8-diacetoxy-4-methylcoumarin, DAMC) and modifies the activity of a number of proteins, thereby influencing cell signaling. To investigate the relationship between CRT expression and radiation response in a human glioma cell line and to evaluate the radiomodifying effects of DAMC. Studies were carried out in an established human glioma cell line (BMG-1) and its isogenic clone overexpressing CRT (CROE, CRT-overexpressing cells) by analyzing clonogenic survival, cell proliferation, micronuclei analysis, and protein levels by Western blotting as parameters of responses. CRT overexpression conferred resistance against radiation-induced cell death in CROE cells (D37 =7.35 Gy, D10 =12.6Gy and D0 =7.25 Gy) as compared to BMG-1 cells (D37 =5.70 Gy, D10 =9.2Gy and D0 =5.6Gy). A lower level of radiation-induced micronuclei formation observed in CROE cells suggested that reduced induction and/or enhanced DNA repair partly contributed to the enhanced radioresistance. Consistent with this suggestion, we noted that CRT-mediated radioresistance was coupled with enhanced grp78 level and reduced P53 activation-mediated prodeath signaling, while no changes were noted in acetylation of histone H4. DAMC-enhanced radiation-induced delayed (secondary) apoptosis, which was higher in CROE cells. CRT overexpression confers resistance against radiation-induced death of human glioma cells, which can be overcome by the polyphenolic acetate DAMC.

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA

BackgroundHER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype.MethodsWe generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets.ResultsTreatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts.ConclusionOur results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Exploiting an Asp-Glu “Switch” in Glycogen Synthase Kinase 3 to Design Paralog Selective Inhibitors for Acute Myeloid Leukemia Treatment

Exploiting an Asp-Glu “Switch” in Glycogen Synthase Kinase 3 to Design Paralog Selective Inhibitors for Acute Myeloid Leukemia Treatment

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Differential methylation EPIC analysis discloses cisplatin-resistance related hypermethylation and tumor-specific heterogeneity within matched primary and metastatic testicular germ cell tumor patient tissue samples

Testicular germ cell tumors (TGCTs) are among the most common solid malignancies in young-adult men, and currently most mortality is due to metastatic disease and emergence of resistance to cisplatin. There is some evidence that increased methylation is one mechanism behind this resistance, stemming from individual studies, but approaches based on matched primary and metastatic patient samples are lacking. Herein, we provide an EPIC array-based study of matched primary and metastatic TGCT samples. Histology was the major determinant of overall methylation pattern, but some clustering of samples related to response to cisplatin was observed. Further differential analysis of patients with the same histological subtype (embryonal carcinoma) disclosed a remarkable increase in net methylation levels (at both promoter and CpG site level) in the patient with cisplatin-resistant disease and poor outcome compared to the patient with complete response to chemotherapy. This further confirms the recent results of another study performed on isogenic clones of sensitive and resistant TGCT cell lines. Differentially methylated promoters among groups of samples were mostly not shared, disclosing heterogeneity in patient tissue samples. Finally, gene ontology analysis of cisplatin-resistant samples indicated enrichment of differentially hypermethylated promoters on pathways related to regulation of immune microenvironment, and enrichment of differentially hypomethylated promoters on pathways related to DNA/chromatin binding and regulation. This data supports not only the use of hypomethylating agents for targeting cisplatin-resistant disease, but also their use in combination with immunotherapies and chromatin remodelers.

Serial transplantation unmasks galectin-9 contribution to tumor immune escape in the MB49 murine model

Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones—either positive or negative for gal-9—from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.

Identifying chromatin features that regulate gene expression distribution

Gene expression variability, differences in the number of mRNA per cell across a population of cells, is ubiquitous across diverse organisms with broad impacts on cellular phenotypes. The role of chromatin in regulating average gene expression has been extensively studied. However, what aspects of the chromatin contribute to gene expression variability is still underexplored. Here we addressed this problem by leveraging chromatin diversity and using a systematic investigation of randomly integrated expression reporters to identify what aspects of chromatin microenvironment contribute to gene expression variability. Using DNA barcoding and split-pool decoding, we created a large library of isogenic reporter clones and identified reporter integration sites in a massive and parallel manner. By mapping our measurements of reporter expression at different genomic loci with multiple epigenetic profiles including the enrichment of transcription factors and the distance to different chromatin states, we identified new factors that impact the regulation of gene expression distributions.

Coordinated Mis-Splicing of Multiple Mitochondrial Iron Metabolism Genes Causes Ring Sideroblast Formation in SF3B1-Mutant MDS

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with >10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant >40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

EttA is likely non-essential in Staphylococcus aureus persistence, fitness or resistance to antibiotics

BackgroundTolerance to antibiotics and persistence are associated with antibiotic treatment failures, chronic-relapsing infections, and emerging antibiotic resistance in various bacteria, including Staphylococcus aureus. Mechanisms of persistence are largely unknown, yet have been linked to physiology under low-ATP conditions and the metabolic-inactive state. EttA is an ATP-binding cassette protein, linked in Eschrechia coli to ribosomal hibernation and fitness in stationary growth phase, yet its role in S. aureus physiology is unknown.ResultsUsing whole genome sequencing (WGS) of serial clinical isolates, we identified an EttA-negative S. aureus mutant (ettAstop), and its isogenic wild-type counterpart. We used these two isogenic clones to investigate the role of ettA in S. aureus physiology in starvation and antibiotic stress, and test its role in persistence and antibiotic tolerance. ettAstop and its WT counterpart were similar in their antibiotic resistance profiles to multiple antibiotics. Population dynamics of ettAstop and the WT were similar in low-nutrient setting, with similar recovery from stationary growth phase or starvation. Supra-bacteriocidal concentration of cefazolin had the same killing effect on ettAstop and WT populations, with no difference in persister formation.ConclusionsLack of ettA does not affect S. aureus antibiotic resistance, beta-lactam tolerance, resilience to starvation or fitness following starvation. We conclude the role of ettA in S. aureus physiology is limited or redundant with another, unidentified gene. WGS of serial clinical isolates may enable investigation of other single genes involved in S. aureus virulence, and specifically persister cell formation.