Common Driver Mutations Research Articles

Development of Novel Murine BRAFV600E-Driven Papillary Thyroid Cancer Cell Lines for Modeling of Disease Progression and Preclinical Evaluation of Therapeutics.

The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a BrafV600E+/-/Pten+/-/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.

Application of the Novel Grading System of Invasive Pulmonary Adenocarcinoma in a Real Diagnostic Scenario: A Brief Report of 9353 Cases

IntroductionThe International Association for the Study of Lung Cancer proposed a novel grading system of invasive pulmonary adenocarcinoma (IPA), but the application of this grading system and its genotypic characterization in the real diagnostic scenario has never been reported. MethodsWe prospectively collected and analyzed the clinicopathological and genotypic features of a cohort of 9353 consecutive patients with resected IPA, including 7134 patients with detection of common driver mutation. ResultsIn the entire cohort, 3 (0.3%) of lepidic, 1207 (19.0%) of acinar, and 126 (23.6%) of papillary predominant IPAs were diagnosed as grade 3. In chronological order, an evident downtrend of the proportion of grade 2 was observed in chronological order. Conversely, the diagnostic ratio of grade 1 (8.0%–14.5%) and grade 3 (27.9%–32.3%) experienced a gradual rise. EGFR mutation was more frequently detected in grade 2 (77.5%) and grade 1 (69.7%) IPA than grade 3 (53.7%, p < 0.001), whereas the mutation rates of KRAS, BRAF, ALK, and ROS1 were higher in grade 3 IPA. More importantly, the rate of EGFR mutation gradually fell as the proportion of high-grade components increased, to 24.3% in IPA with more than 90% high-grade components. ConclusionsThe grading system for IPA could be applied to stratify patients with different clinicopathological and genotypic features in a real diagnostic scenario.

Whole-genome characterization of large-cell lung carcinoma: A comparative analysis based on the histological classification.

Background: According to the 2015 World Health Organization classification, large cell neuroendocrine carcinoma (LCNEC) was isolated from Large-cell lung cancer (LCLC) tumors, which constitutes 2%-3% of non-small cell lung cancer (NSCLC). However, LCLC tumors are still fairly vaguely defined at the molecular level compared to other subgroups. Materials and Methods: In this study, whole-genome sequencing (WGS) was performed on 23 LCLC and 15 LCNEC tumor specimens. Meanwhile, data from the TCGA (586 LUADs and 511 LUSCs) and U Cologne (120 SCLCs) were analyzed and compared. Results: The most common driver mutations were found in TP53 (13/23, 57%), FAM135B (8/23, 35%) and FAT3 (7/23, 30%) in LCLC, while their counterparts in LCNEC were TP53 (13/15, 87%), LRP1B (6/15, 40%) and FAT1 (6/15, 40%). Notably, FAM135B mutations only occurred in LCLC (P = 0.013). Cosmic signature analysis revealed widespread defective DNA mismatch repair and tobacco-induced mutations in both LCLC and LCNEC. Additionally, LCNEC had a higher incidence of chromosomal copy number variations (CNVs) and structural variations (SVs) compared with LCLC, although the differences were not statistically significant. Particularly, chromothripsis SVs was significantly associated with CNVs. Furthermore, mutational landscape of different subtypes indicated differences between subtypes, and there seems to be more commonalty between our cohort and SCLC than with other subtypes. SMARCA4 mutations may be specific driver gene alteration in our cohort. Conclusion: Our results support that LCLC and LCNEC tumors follow distinct tumorigenic pathways. To our knowledge, this is the first genome-wide profiling comparison of LCLC and LCNEC.

Oncogenic BRAFV600E induces microglial proliferation through extracellular signal-regulated kinase and neuronal death through c-Jun N-terminal kinase.

Activating V600E in v-Raf murine sarcoma viral oncogene homolog B (BRAF) is a common driver mutation in cancers of multiple tissue origins, including melanoma and glioma. BRAFV600E has also been implicated in neurodegeneration. The present study aims to characterize BRAFV600E during cell death and proliferation of three major cell types of the central nervous system: neurons, astrocytes, and microglia. Multiple primary cultures (primary cortical mixed culture) and cell lines of glial cells (BV2) and neurons (SH-SY5Y) were employed. BRAFV600E and BRAFWT expression was mediated by lentivirus or retrovirus. Blockage of downstream effectors (extracellular signal-regulated kinase 1/2 and JNK1/2) were achieved by siRNA. In astrocytes and microglia, BRAFV600E induces cell proliferation, and the proliferative effect in microglia is mediated by activated extracellular signal-regulated kinase, but not c-Jun N-terminal kinase. Conditioned medium from BRAFV600E-expressing microglia induced neuronal death. In neuronal cells, BRAFV600E directly induces neuronal death, through c-Jun N-terminal kinase but not extracellular signal-regulated kinase. We further show that BRAF-related genes are enriched in pathways in patients with Parkinson's disease. Our study identifies distinct consequences mediated by distinct downstream effectors in dividing glial cells and in neurons following the same BRAF mutational activation and a causal link between BRAF-activated microglia and neuronal cell death that does not require physical proximity. It provides insight into a possibly important role of BRAF in neurodegeneration as a result of either dysregulated BRAF in neurons or its impact on glial cells.

S-55-4: HISTOPATHOLOGY-GENOTYPE CORRELATION OF ALDOSTERONE- PRODUCING ADENOMAS

Primary aldosteronism (PA) is a vastly underdiagnosed (&lt;1 in 500) cause of secondary hypertension in which aldosterone production is inappropriately high relative to suppressed plasma renin levels. This is of major concern as untreated patients are at increased risk for cardiovascular diseases, stroke, and other comorbidities even when compared to matched essential hypertension patients. PA is commonly caused sporadically by aldosterone producing adenomas (APAs) that harbors somatic mutations in aldosterone driver genes. It can account for up to 5 to 10% of hypertensive cases or up to 10 to 20% of resistant hypertensive cases. The current recommended treatment for APAs when only affecting one adrenal is a unilateral adrenalectomy. This has allowed the excised tissue to be extensively studied and characterized. Interestingly APAs have been found to occur with a spectrum of cell morphology and clinical phenotype. This heterogeneity seem to have a genetic basis. KCNJ5 mutant APAs, the most common genotype of APAs, have zona fasciculata (ZF) like cell morphology &amp; secrete hybrid steroids. They commonly are large and frequently occur among young females and Oriental patients. Whereas CACNA1D mutant APAs are smaller, more zona glomerulosa (ZG) like (i.e. more compact than ZF), and more common in Black patients. To note, CACNA1D is also the most common aldosterone driver mutations in aldosterone producing micronodules and elderly patients, followed by aldosterone driver mutations in ATP1A1 and ATP2B3. CTNNB1 mutant APAs that also harbor GNA11/GNAQ mutations have cells that seem to dedifferentiate towards the adrenal-gonadal precursor cell type as they express the LH/HCG receptor ten fold more than other mutant APAs or even CTNNB1 mutant only APAs. This would explain why patients with the double mutations often present at times of high LH/HCG. These distinct genotype specific phenotypes could be used to facilitate screening and diagnosis of PA. For example, hybrid steroid measurements could be used for screening of KCNJ5 mutant APAs; or onset of hypertension in the first trimester (the period of peak HCG secretion), compared to pregnancy associated hypertension in later trimesters, should prompt consideration for a double mutant APA. Moreover, identification of these somatic mutations has already led to potential therapeutic treatments. For example, chemical screens have identified macrolides that selectively inhibit mutant KCNJ5. Thus, histopathology genotype correlation of APAs have provided new opportunities for the screening, diagnosis, management, and treatment of PA. Further elucidations of the cellular mechanisms associated with each correlation will help to identify the pathological and physiological pathways, and thus novel therapeutic targets.

Multi-institution analysis of tumor mutational burden and outcomes in pediatric central nervous system tumor patients.

Pediatric central nervous system (CNS) tumors are the leading cause of pediatric cancer mortality. Research addressing genomic biomarkers and clinical outcomes is needed to inform therapeutic decision-making. We conducted a retrospective analysis of pediatric patients (age <21) diagnosed with a primary CNS tumor at four upstate New York hospitals from 2008 to 2021. Clinical and histopathologic data were identified from each patient, including genomic analysis of somatic mutations and tumor mutational burden (TMB) where available. These variables were each compared with overall survival using Cox regression analyses. Multivariable analysis was conducted to identify patient characteristics that may independently predict survival. We identified 119 patients. Common tumor types included low-grade glioma (N=51), high-grade glioma (N=29), and medulloblastoma (N=11). Common driver mutations included TP53 inactivation (N=16), BRAF-KIAA1549 fusion (N=16), FGFR1 amplification (N=12), BRAF V600E mutation (N=12), NF1 loss (N=12), and H3F3A K28M mutation (N=6). Median TMB was one mutation/megabase (mut/Mb, range=0-132). Overall survival was 79.9%. Variables associated with poorer survival on univariable analysis were higher TMB (p=.002, HR 4.97), high-grade tumors (p=.009, HR 84.3), and high-grade glioma histology (p=.021, HR 3.14). Multivariable analyses further identified TMB (p=.011, HR 4.46) and high-grade histology (p=.015, HR 5.28) as independently predictive of worse survival. Tumor progression was more common in high-TMB (N=15, 44%) than in low-TMB tumors (N=19, 35%). High TMB is correlated with higher rates of progression and death as compared to low-TMB tumors. These findings may help identify patients who may benefit from alternative treatments, such as immunotherapies.

Discrepancies association between FLT3 inhibitors use and prognosis of acute myeloid leukemia: systematic review and meta-analysis

Acute myeloid leukemia (AML) is a malignancy of proliferative, clonal, abnormally, or poorly differentiated cells of the hematopoietic system, characterized by clonal evolution and genetic heterogeneity. Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of all AML cases, with the internal tandem duplication (ITD) representing the most common type of FLT3 mutation. FLT3-ITD is a common driver mutation that presents with a high leukemic burden and confers a poor prognosis in patients with AML. In this systematic review and meta-analysis, we present a detailed review of current clinical evidence of FLT3 inhibitors and their use in AML, and discrepancies association between FLT3 inhibitors use and prognosis of acute myeloid leukemia and maintenance setting.

A pilot study to evaluate tissue- and plasma-based DNA driver mutations in a cohort of patients with pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions to pancreatic ductal adenocarcinoma that are challenging to manage due to limited imaging, cytologic, and molecular markers that accurately classify lesions, grade of dysplasia, or focus of invasion preoperatively. The objective of this pilot study was to determine the frequency and type of DNA mutations in a cohort of surgically resected, pathologically confirmed IPMN, and to determine if concordant mutations are detectable in paired pretreatment plasma samples. Formalin-fixed paraffin-embedded (FFPE) tissue from 46 surgically resected IPMNs (31 low-grade, 15 high-grade) and paired plasma from a subset of 15 IPMN cases (10 low-grade, 5 high-grade) were subjected to targeted mutation analysis using a QIAseq Targeted DNA Custom Panel. Common driver mutations were detected in FFPE from 44 of 46 (95.6%) IPMN cases spanning all grades; the most common DNA mutations included: KRAS (80%), RNF43 (24%), and GNAS (43%). Of note, we observed a significant increase in the frequency of RNF43 mutations from low-grade to high-grade IPMNs associated or concomitant with invasive carcinoma (trend test, P = 0.01). Among the subset of cases with paired plasma, driver mutations identified in the IPMNs were not detected in circulation. Overall, our results indicate that mutational burden for IPMNs is a common occurrence, even in low-grade IPMNs. Furthermore, although blood-based biopsies are an attractive, noninvasive method for detecting somatic DNA mutations, the QIAseq panel was not sensitive enough to detect driver mutations that existed in IPMN tissue using paired plasma in the volume we were able to retrieve for this retrospective study.

A novel combination treatment of antiADAM17 antibody and erlotinib to overcome acquired drug resistance in non-small cell lung cancer through the FOXO3a/FOXM1 axis.

After the identification of specific epidermal growth factor receptor (EGFR)-activating mutations as one of the most common oncogenic driver mutations in non-small cell lung cancer (NSCLC), several EGFR-tyrosine kinase inhibitors (EGFR-TKIs) with different clinical efficacies have been approved by various health authorities in the last two decades in targeting NSCLC harboring specific EGFR-activating mutations. However, most patients whose tumor initially responded to the first-generation EGFR-TKI developed acquired resistance. In this study, we developed a novel combination strategy, "antiADAM17 antibody A9(B8) + EGFR-TKIs", to enhance the efficacy of EGFR-TKIs. The addition of A9(B8) was shown to restore the effectiveness of erlotinib and overcome acquired resistance. We found that when A9(B8) antibody was treated with erlotinib or gefitinib, the combination treatment synergistically increased apoptosis in an NSCLC cell line and inhibited tumor growth in vivo. Interestingly, the addition of A9(B8) could only reduce the survival of the erlotinib-resistant NSCLC cell line and inhibit the growth of erlotinib-resistant tumors in vivo but not gefitinib-resistant cells. Furthermore, we revealed that A9(B8) overcame erlotinib resistance through the FOXO3a/FOXM1 axis and arrested the cell cycle at the G1/S phase, resulting in the apoptosis of cancer cells. Hence, this study establishes a novel, promising strategy for overcoming acquired resistance to erlotinib through the FOXO3a/FOXM1 axis by arresting the cell cycle at the G1/S phase.

Abstract PR004: In vitro and in vivo CRISPR screens in colorectal cancer organoids identify suppressors of metastasis

Abstract Colorectal cancer (CRC) is the 2nd-leading cause of cancer-related deaths worldwide and metastatic disease remains a major unmet clinical need. Nevertheless, the molecular underpinnings of metastatic spread and colonization of distant sites remain elusive. To systematically identify functional genes contributing to metastasis in a physiologically relevant model of CRC, we developed a novel platform enabling robust, pooled CRISPR/Cas9 screening in primary colonic organoids engineered with common CRC driver mutations in Apc, Kras, and Tp53. We performed parallel in vitro invasion and in vivo metastasis gain-of-function CRISPR/Cas9 knockout screens with high-content gRNA libraries to uncover novel suppressors of metastasis. We confirmed resulting hits as bona-fide metastatic suppressors using both pooled validation screens and single-gene approaches. Genes scoring in both in vitro and in vivo screens included previously identified regulators of migration and invasion, such as Ctnna1, and genes scoring only in the in vivo screen included novel negative regulators of metastasis such as Bcl2l13. These hits in the murine system were supported by human TCGA data, indicating that low expression of these genes is associated with worse 5-year survival in colon and rectal adenocarcinoma patients (COAD and READ). Utilizing transwell and live-imaging assays, we found that sgRNAs targeting Ctnna1 markedly enhanced invasion and migration of CRC organoids while sgRNAs targeting Bcl2l13 had no effect, an observation consistent with our in vitro screen. Therefore, while loss of Ctnna1 enhances metastasis by augmenting invasion and migration, loss of Bcl2l13 acts through alternative mechanisms. Single cell RNA sequencing of primary and metastatic lesions derived from Bcl2l13 knockout and control CRC tumors identified novel putative pathways promoting metastatic spread via migration-independent mechanisms, including upregulation of hypoxia signalling and recruitment of pro-tumorigenic macrophages. Thus, our CRISPR/Cas9 screening pipeline is a viable approach for the identification of novel regulators of CRC metastasis in vitro and in vivo, uncovering both motility and migration-dependent and independent paths to metastatic success. These findings will help untangle the complex web of molecular events underlying CRC metastasis and provide clinically-relevant insights into the process of human CRC metastasis. Citation Format: Zvi Cramer, Xin Wang, Nicolae Adrian Leu, Keara Monaghan, Joshua H. Rhoades, Yuhua Tian, Joshua Rico, Diego Mendez, Ricardo Petroni, Melissa S. Kim, Ning Li, Christopher J. Lengner, M. Andres Blanco. In vitro and in vivo CRISPR screens in colorectal cancer organoids identify suppressors of metastasis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR004.

FLT3ITD drives context-specific changes in cell identity and variable interferon dependence during AML initiation

AbstractAcute myeloid leukemia (AML) initiation requires multiple rate-limiting mutations to cooperatively reprogram progenitor cell identity. For example, FLT3 internal tandem duplication (FLT3ITD) mutations cooperate with a variety of different initiating mutations to reprogram myeloid progenitor fate. These initiating mutations often skew toward either pediatric or adult AML patient populations, though FLT3ITD itself occurs at similar frequencies in both age groups. This raises the question of whether FLT3ITD might induce distinct transcriptional programs and unmask distinct therapeutic vulnerabilities when paired with pediatric, as opposed to adult AML-initiating mutations. To explore this possibility, we compared AML evolution in mice that carried Flt3ITD/NUP98-HOXD13 (NHD13) or Flt3ITD/Runx1DEL mutation pairs, which are respectively most common in pediatric and adult AML. Single-cell analyses and epigenome profiling revealed distinct interactions between Flt3ITD and its cooperating mutations. Whereas Flt3ITD and Flt3ITD/Runx1DEL caused aberrant expansion of myeloid progenitors, Flt3ITD/NHD13 drove the emergence of a pre-AML population that did not resemble normal hematopoietic progenitors. Differences between Flt3ITD/Runx1DEL and Flt3ITD/NHD13 cooperative target gene expression extended to fully transformed AML as well. Flt3ITD/NHD13 cooperative target genes were enriched in human NUP98-translocated AML. Flt3ITD/NHD13 selectively hijacked type I interferon signaling to drive expansion of the pre-AML population. Blocking interferon signaling delayed AML initiation and extended survival. Thus, common AML driver mutations, such as FLT3ITD, can coopt different mechanisms of transformation in different genetic contexts. Furthermore, pediatric-biased NUP98 fusions convey actionable interferon dependence.

The Progress of Growth and Apoptosis Are Associated with Both of AURKA and Aurkb in Marimo Cells with Calr Gene Mutation

Objective: Mutations of calreticulin (CALR) are a breakthrough discovery in exon 9 as most common driver mutations in janus kinase 2 (JAK2) or thrombopoietin receptor (MPL)-negative mutant essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Several new inhibitors have displayed a relatively good therapeutic prospect after the JAK inhibitors, but still not completely cure the patients on their own. Thus, improvement of the therapeutic effect of these new inhibitors is particularly important through exploration of the related targets, which is probably compensated the function of original target in patients. Methods: The protein level of aurora was detected in CALR mutant patients, and was quantified by calculating the gray value of WB band. The biological function was examined in CALR mutant cell line (MARIMO) after aurora inhibition by EdU staining, colony assay, western blot, Annexin V, and flow cytometry assay, respectively. Transcriptomic analyzed the enriched genes and related signaling pathway in MARIMO cells with knockdown of Aurora A (AURKA) or Aurora B (AURKB). Results: Here, we found that Aurora B, similarly with Aurora A, was activated in CALR mutant patients. Selective inhibition of AURKA with MLN 8237 induced cell-growth arrest, maturation and apoptosis, and this cell phenotype was further enhanced when the AURKB was also inhibited as the increase of inhibitors' concentration. Specific downregulation of AURKB alone showed obvious inhibition on cell growth, and produced a similar effect on CALR mutant cells treated with MLN 8237 or interfered AURKA. Transcriptomic analyses revealed a replication reaction on cells caused by knockdown of AURKA or AURKB, which also induced a similar inhibition for mitogen-activated protein kinase (MAPK) signaling, but activation for metabolic and apoptosis pathway. Conclusion: These results suggested that both of AURKA and AURKB were associated with the progress of growth and apoptosis in MARIMO cells with CALR gene mutation, and provided a reference for recently proposed complementary therapies with Aurora kinase inhibitors in CALR mutated MPNs.

Abstract B040: In silico drug design using KRASG12C as a theranostic target for pancreatic cancer

Abstract Pancreatic cancer is one of the most lethal malignancies worldwide, it often results in therapeutic failure and unfavorable outcome because of the absence of screening strategies, difficulties in tumor early detection due to the lack of specific signs/symptoms and aggressive behavior of the disease. Early detection of this cancer is a challenge with the technology we have, its late detection results in a poor outcome and reduced quality of life for the patient. The treatment success depends on surgical intervention, which is not always possible. Thus, trying to find alterations that occur in the early stages of pancreatic cancer is needed. Taking into consideration that the accumulation of genetic and epigenetic alterations results in a complex and heterogeneous disease, each patient has a unique tumor. The heterogeneity of cancer is not restricted to inter-patient differences but, intra-tumoral heterogeneity contributes to functional properties of tumor cell subclones, including proliferative capacity and progression to aggressive phenotypes such as drug tolerance and metastatic potential. Driver mutations are responsible for malignant transformation and tumor progression across different cancer types. One of the most common driver mutations are those leading to the constitutive activation of KRAS oncogene, a GTPase enzyme responsible for cell proliferation and survival that is frequently mutated in pancreatic cancer. Gain-of-function mutation of KRAS gene leads to a protein constitutively activated, which stimulates the proliferation of tumor cells. The c.34G&amp;gt;T missense mutation (rs121913530) causes the substitution of glycine for cysteine in the encoded protein KRASG12C; this mutation has been related to poorer prognosis and therapy resistance in lung cancer. As KRAS occurs before the progression of pancreatic cancer to invasive carcinoma, it is a promising target for new biomarker development. We design three near-infrared probe-drug conjugate directed to the KRASG12C and to predict in silico the best orientation and conformation of the test compounds bound to the KRAS G12C (PDB code: 6OIM), the PyRx program was used, which uses AutoDock Vina as docking software. Based on these profiles and structures of the compounds, AutoDock Vina infers the possible pharmacological interactions and allows the most promising compounds to be classified according to the studied biological activity, using the empirical score function for this classification. The AMG 510 (established inhibitor) and GDP (endogenous activator) were used as a reference to establish the docking box coordinates. The binding energy of the three compounds predicted by docking was analyzed and classified according to the binding affinity described in Kcal/mol. The tested compounds show an excellent affinity for this protein based on the binding energy results obtained by docking using the crystallized KRAS G12C protein. Therefore, they could be great candidates for further chemical synthesis studies aiming to improve diagnosis, treatment, and patient survival in pancreatic cancer. Citation Format: Barbara M. Barbosa, Allana C. F. Martins, Roberto S. Gomes, Claudia A. Rainho. In silico drug design using KRASG12C as a theranostic target for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B040.

Interferon-alpha2 Treatment Reduces Circulating Neutrophil Extracellular Trap Levels in Myeloproliferative Neoplasms

Background and aim Arterial and venous thrombosis are major life-threatening events for patients with Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Neutrophil activation and, in particular, the formation of neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of MPNs and thrombosis, particularly in patients with the most common MPN phenotypic driver mutation, JAK2V617F. Moreover, ruxolitinib treatment reduces ex vivo NET formation in JAK2V617F-positive patients with MPNs. In this study, we aimed to clarify the effect of interferon alpha (IFNα) and hydroxyurea (HU) treatment on the levels of circulating NETs in patients with MPNs. Methods Serum NET (myeloperoxidase-DNA complexes) levels were measured using an in-house ELISA in pre-treatment samples collected from 128 patients with MPNs enrolled in the DALIAH trial (NCT01387763), a randomized controlled phase 3 clinical study. NETs were likewise quantified in 85 paired samples collected after 12 months of therapy with HU or either of two different formulations of pegylated IFNα (PEG-IFNα2a or PEG-IFNα2b). Results At baseline, NET levels did not vary significantly between patients with different MPN sub-diagnoses (ET: 48, PV: 61, Pre-MF: 10, PMF: 9), phenotypic driver mutations (JAK2V617F, CALR, MPL, or triple negative), or between those with or without a history of major thrombosis. However, patients with PV and a JAK2V617F allele burden ≥ 50% at baseline had significantly higher baseline NET levels than those with an allele burden < 50% (p=0.006). NET levels at baseline correlated significantly with neutrophil count (r=0.29, p=0.001), neutrophil-to-lymphocyte ratio (NLR)(r=0.26, p=0.004) and JAK2V617F allele burden (r=0.22, p=0.03), particularly in patients with PV and allele burden ≥ 50% (r=0.50, p=0.01, r=0.56, p=0.007 and r=0.45, p=0.03 respectively), while the same was not observed at follow-up. Furthermore, after 12 months of treatment, NET levels decreased in 70% of patients, with an average decrease of 48%, compared to baseline levels. The effect of treatment was particularly pronounced in patients with PV with an allele burden ≥ 50%, of whom 76% showed decreased NET levels, with an average decrease of 60%, while 67% of patients with allele burden < 50% showed an average reduction in NET levels of only 36% (Figure 1). While 77% and 73% of patients treated with PEG-IFNα-2a or PEG-IFNα-2b, respectively, had decreased NET levels, the same applied to only 53% of patients treated with HU (Figure 2). Discussion and conclusion The increased NET levels in patients with PV with a JAK2V617F allele burden ≥ 50% indicate that the JAK/STAT pathway is involved in NET formation and/or clearance. In keeping with this, the highest proportion of patients with decreased NET levels after treatment and the highest average decrease were seen in patients with PV and a baseline JAK2V617F allele burden ≥ 50%. NET levels at baseline correlated with neutrophil count and NLR, however normalization of blood counts alone did not account for the observed decrease in NET levels. Accordingly, treatment with IFNα, which has previously been shown to target the JAK2V617F clone preferentially, effectively reduced circulating NET levels, and to a greater extent than HU treatment. This may relate to our previous finding that three months' IFNα2 treatment downregulates the expression of PAD4, which is implicated in NET formation. Although, we found no difference in NET levels in patients with and without a history of major thrombotic events, these data suggest that IFNα2 treatment, by reducing levels of potentially prothrombotic NET, may contribute to reduced risk of this complication in patients with MPNs, and that it is an advantageous treatment compared to HU in this respect. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK2 V617F Mutation and Associated Chromosomal Alterations Involving JAK2 in Pre-HCT Blood Samples and Transplant Outcomes in Myelofibrosis Subtypes

Introduction: Myelofibrosis (MF) is a BCR-ABL1-negative myeloproliferative neoplasm that develops either de novo (primary myelofibrosis; PMF) or secondary to polycythemia vera (post-PV) or essential thrombocythemia (post-ET). JAK2 V617F is the most common somatic driver mutation in MF known to associate with disease progression. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment modality for MF. There are limited data regarding the prognostic utility of JAK2 V617F mutation and mosaic chromosomal alterations (mCAs) spanning JAK2 in MF patients undergoing HCT. Methods: The study included 924 patients undergoing HCT for myelofibrosis (634 PMF, 135 post-PV, and 155 post-ET) between 2000-2016. Pre-HCT peripheral blood samples, clinical and post-HCT data were provided by the Center for International Blood and Marrow Transplant Research (CIBMTR) registry and biorepository. We used PacBio Single Molecule Real-Time (SMRT) sequencing to detect the JAK2 V617F mutation, and Illumina Infinium Global Screening Array-24v1-0 to identify mCAs; this analysis focused on mCAs involving JAK2 (9p24.1). We used the Kaplan-Meier estimator to calculate the probability of overall survival (OS), and the cumulative incidence estimator for relapse/disease progression incidence and non-relapse mortality (NRM), where each was treated as a competing event for the other. Cox proportional hazard models were used for multivariable analyses. Analyses were completed separately by MF disease subtype. Follow up started at date of HCT. Results:JAK2 V617F mutation was detected in 562 patients (60.8%) with noted expected differences by disease subtype (57.6% of patients with PMF, 97% of post-PV, and 42.6% of post-ET). Almost all patients with mCAs involving JAK2 region were found to be JAK2 V617F mutation-positive (366 out of 374, 97.9%). In JAK2 V617F positive cases, the frequencies of mCA-positive patients per MF subtypes were as follow: 58.08% of PMF, 89.3% of post-PV, and 56.06% of post-ET. The mean allele burden for JAK2 V617F mutation was 57%, 78%, and 60% in PMF, post-PV, and post-ET, respectively. The probabilities of 3-year post-HCT OS were 55% (95% CI=51-58) for PMF, 59% (95% CI=51-67) for post-PV and 59% (51-66) for post-ET. The corresponding 3-year post-HCT cumulative incidences for relapse were 31% (95% CI=28-35), 31% (95% CI=24-40), and 33% (95% CI=26-41); and for NRM were 29% (95% CI=26-33), 25% (95% CI=18-32), and 23% (95% CI=17-30), for PMF, post-PV, and post-ET, respectively. In PMF, JAK2V617F mutation, JAK2 mCAs, or mutation allele burden were not associated with any of the evaluated HCT outcomes (p>0.05 for all). In post-PV, all four patients without JAK2V617F mutation died in the first 12 months after transplantation. No statistically significant associations between HCT outcomes and JAK2V617F allele burden was noted (p>0.05). For post-ET, patients with high mutation allele burden (³60%) had three-fold excess risk of NRM compared with mutation-negative patients (hazard ratio [HR]=2.9, 95% CI=1.21-6.93, p=0.01). No risk difference was noted between post-ET patients with low allele burden and risk of NRM (HR=0.89, 95% CI=0.34-2.31). No statistically significant association between JAK2V617F mutation status and risk of relapse for high or low mutation allele burden as compared with mutation-negative post ET patients was observed (HR=1.42, p=0.30 in patients with allele burden<60%, and HR= 1.54, p=0.26 for patients with allele burden³60%). JAK2V617F mutation status in post-ET patients was not associated with post HCT OS (HR=1.31, 95% CI=0.78-2.2, p=0.31). Conclusion: The effect of JAK2V617F mutation on post-HCT outcome differs by myelofibrosis subtypes. HCT outcomes in primary and post-PV myelofibrosis were not associated with JAK2 alterations, but high JAK2V617F mutation allele burden in post-ET was associated with elevated risk of non-relapse mortality but did not translate to a statistically significant survival disadvantage. Our findings suggest that allogeneic HCT for myelofibrosis may alleviate the known negative effect of JAK2V617F mutation in those patients, particularly for primary myelofibrosis and post-PV. It is essential to consider evaluating these findings within the context of other MF driver genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Analysis of Human Clonal Hematopoiesis Identifies Distinct Impact of DNMT3A and TET2 mutations on Hematopoietic Differentiation

Clonal hematopoiesis (CH) increases the risk of hematological malignancies and other adverse outcomes. The most common driver mutations occur in DNMT3A and TET2. However, it is still unclear how mutant DNMT3A and TET2 perturb human stem/progenitor cell homeostasis allowing mutant clones to expand. Studies of human CH have been limited by availability of samples from subjects without co-existing malignancy and high-precision methods for comparing mutant and wild-type (WT) cells in the same individual. To address this gap, we screened 195 bone marrow (BM) samples collected from subjects undergoing hip replacement surgery for CH by targeted DNA sequencing. As DNMT3A and TET2 regulate DNA methylation and gene expression, we employed multiomic single-cell analysis with TARGET-seq to combine single-cell genotyping with transcriptome sequencing on FACS index sorted cells. We developed an improved method, TARGET-seq+, which provides greater sensitivity transcript detection, resulting in a median of 4500 genes/cell with high-sensitivity genotyping (95% of cells genotyped; 7-11% allele dropout rate). We performed TARGET-seq+ on 12,942 BM stem, progenitor and mature cells from 9 CH samples with predicted loss-of-function mutations (4 single mutation cases & 5 multiple mutation cases) and 3 normal controls (Fig. 1). Of these, 87% of cells passed quality controls and were successfully genotyped. Integrated transcriptomic and genotyping analysis showed DNMT3Amut and TET2mut cells occupy a common differentiation trajectory with WT cells. Within DNMT3A-mutant samples, the mutant clone constituted 5-70% of transcriptomic and immunophenotypic long-term hematopoietic stem cells (LT-HSC). There was a further 1.5 to 1.7-fold expansion of DNMT3Amut cells in early erythro-megakaryocytic multipotent progenitors and lympho-myeloid multipotent progenitors (LMPP) compared to LT-HSC. Across most samples, clone size was maintained throughout differentiation with no evidence of differentiation delay or lineage bias. Differential expression analysis revealed subtle cell type-specific differences between WT and mutant cells, with upregulation of megakaryocytic lineage markers in mutant LMPP and downregulation of erythroid lineage markers in mutant erythroid progenitors. TET2mut clones were also identified in LT-HSCs at a clone size of 1-21% but expanded further in multiple downstream progenitors. Maximal expansion (2-12 fold) was seen in the myeloid lineage, but clones also expanded in the erythroid, megakaryocytic and lymphoid lineages (mean clone size 2 to 5-fold larger than in LT-HSC). Comparison of mutant and WT granulocyte-monocyte progenitors (GMP) identified upregulation of myeloid lineage-affiliated transcription factors, (e.g. CEBPD and IRF8) in TET2mut cells. Increased expression of myeloid gene signatures was also seen within TET2mut HSC and LMPP, suggesting myeloid lineage bias arises early in differentiation. Concordantly, TET2mut HSC were shifted towards a more differentiated state in pseudotime and downregulated LT-HSC signatures. In one sample with biallelic TET2 mutations, the TET2 single and double mutant clones were each detected in only 1 out of 109 (0.9%) CD90+CD49f+ LT-HSCs. However, the double mutant clone expanded dramatically in the myeloid lineage, contributing to >50% of cells within GMP, dendritic cell progenitor and neutrophil/monocyte precursor compartments, consistent with frequent detection of biallelic TET2 mutations in myeloid leukemias. Comparison of gene expression between differentiation stage-matched WT cells in CH samples and in age-matched normal controls allowed us to identify the impact of CH clones on WT cells, co-existing within the same BM micro-environment. WT HSCs in DNMT3A and TET2 mutant samples showed enhanced expression of inflammatory, quiescence and chemokine gene signatures compared to WT HSCs in non-CH samples. In summary, we present the first detailed analysis of the impact of DNMT3A and TET2 mutations on hematopoiesis in humans with unperturbed CH. The selective advantage of DNMT3A-mutant clones occurs primarily at the level of HSC and early progenitors, whereas TET2-mutant clones expand not only in HSC but also further throughout differentiation (Fig. 2). There is a non-cell autonomous impact on WT HSCs in CH. Our work provides a foundation for understanding the mechanistic basis of pre-leukemic clonal expansion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Erythroid and Megakaryocytic Differentiation Program in JAK2-Mutated Acute Myeloid Leukemia with or without Antecedent Myeloproliferative Neoplasm

JAK2 V617F is the most common driver mutation for Ph-negative MPN and related blast transformation (MPN-BP). In contrast, JAK2 V617F is rare (<5%) in acute myeloid leukemia (AML) lacking an antecedent MPN (nonMPN AML). JAK2 V617F skews the differentiation of hematopoietic stem/progenitor cells (HSPC) towards erythroid and megakaryocytic lineages and in conjunction with TP53 deficiency/mutations results in acute erythroid leukemia in mice (Rampal et al., PNAS 2014). These phenotypes, although also reported in patients with MPN-BP (Mesa et al., Blood 2005), remain incompletely understood. In this study, we set out to study these phenotypes at the protein, epigenomic and transcriptomic levels in JAK2-mutated AML comparing MPN-BP to nonMPN AML. The study cohort included 58 MPN-BP (16 PV, 16 ET, 14 PMF and 12 MPN-U) and 27 nonMPN AML patients (11 de novo AML, 12 AML-MRC, 4 t-AML) with JAK2 V617F mutation (Table 1). In addition, 100 patients with JAK2 WT AML were also included as control group. The median age at diagnosis in MPN-BP and nonMPN AML subgroups as well as in JAK2 WT AML was similar (70 years vs 71 years vs 70 years, respectively) and showed a male preponderance. The median interval between MPN diagnosis and blast transformation was 4.3 years (range, 0-20.5 years). Overall survival was inferior in MPN-BP compared to nonMPN AML (median survival: 7.4 vs 19.3 months). MPN-BP showed a higher rate of MF-2 or 3 myelofibrosis (78% vs 33%, p<0.05), osteosclerosis (50% vs 15%, p<0.05) and marrow necrosis (7% vs 0%) on bone marrow biopsies than nonMPN AML. On immunohistochemistry, 28% (10/32) MPN-BP cases (vs 0% nonMPN AML, p<0.05) were classified as acute leukemia with erythroid/megakaryocytic differentiation (M6b and/or M7) based on expression of E-cadherin, CD71, Glycophorin A, and CD61 on leukemic blasts. ERG, FLI1, and LMO2, transcription factors that are often expressed in HSPCs, were also expressed in leukemic blasts of MPN-BP and nonMPN AML. Aberrant TP53 expression by immunohistochemistry was seen in 31% of MPN-BP (5/9 and 5/21 cases with and without erythroid/megakaryocytic differentiation, respectively) and 17% (3/18) of nonMPN AML. The most common mutations on next-generation sequencing included TP53, RUNX1, ASXL1, TET2, DNMT3A, SRSF2, IDH1, IDH2 and EZH2 in each group. However, of these, TP53 mutations were more frequent in MPN-BP (34% vs 11% in nonMPN AML, p<0.05) and TET2 mutations more frequent in nonMPN AML (48% vs 24% in MPN-BP, p<0.05). Interestingly, NPM1 and FLT3, which are among the most mutated genes in AML were not detected in either MPN-BP or nonMPN AML. Based on the over-representation of erythroid/megakaryocytic phenotype in MPN-BP seen on immunohistochemistry, we hypothesized that MPN-BP blasts might exhibit an upregulated epigenetic and transcriptional program involved in erythroid/megakaryocytic differentiation. RNA-seq and ATAC-seq analysis were performed on flow cytometry sorted CD34+ leukemic blast populations from 11 MPN-BP and 6 nonMPN AML, as well as similar sorted populations from normal controls and JAK2 WT AML. Principal component analysis showed overlapping MPN-BP and nonMPN AML samples, both of which were separated from JAK2 WT AML. Differential expression analysis confirmed nearly identical gene expression between blasts from MPN-BP and nonMPN AML. In addition, consistent with findings by immunohistochemistry, the expression of genes specifically involved in erythroid/megakaryocytic differentiation (TFRC, GYPA, HBB, SPTA1, EPOR, ITGA2B, ITGB3, PF4, ALAS2, GP1BA, ENG and MPL) were upregulated in MPN-BP. Hierarchical clustering using Ward's method on both RNA-seq and ATAC-seq normalized counts revealed that the JAK2 WT AML cluster was distinct from the MPN-BP and nonMPN AML cluster (Figure 1). Lineage deconvolution of ATAC-seq using hematopoietic differentiation stages (Glass et al., Blood 2017, Dilip et al., Blood 2021) to evaluate lineage characteristics of the blasts showed hematopoietic stem cell (HSC) predominance in JAK2 WT AML while MPN-BP and nonMPN AML displayed enrichment of multipotent progenitor (MPP), megakaryocyte-erythrocyte progenitor (MEP) and erythroid signatures. Collectively, our data demonstrates enrichment of erythroid and megakaryocytic programs in both JAK2-mutated MPN-BP and JAK2-mutated nonMPN AML, suggesting that chromatin accessibility and transcription may be driven by mutated JAK2 regardless of antecedent MPN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Uterine lavage identifies cancer mutations and increased TP53 somatic mutation burden in individuals with ovarian cancer.

Current screening methods for ovarian cancer (OC) have failed to demonstrate a significant reduction in mortality. Uterine lavage combined with TP53 ultra-deep sequencing for the detection of disseminated OC cells has emerged as a promising tool, but this approach has not been tested for early-stage disease or non-serous histologies. In addition, lavages carry multiple background mutations, the significance of which is poorly understood. Uterine lavage was collected preoperatively in 34 patients undergoing surgery for suspected ovarian malignancy including 14 patients with benign disease and 20 patients with OC (6 non-serous and 14 high grade serous-like (serous)). Ultra-deep duplex sequencing (~3000x) with a panel of common OC genes identified the tumor mutation in 33% of non-serous (all early stage) and in 79% of serous cancers (including four early stage). In addition, all lavages carried multiple somatic mutations (average of 25 mutations per lavage), more than half of which corresponded to common cancer driver mutations. Driver mutations in KRAS, PIK3CA, PTEN, PPP2R1A and ARID1A presented as larger clones than non-driver mutations and with similar frequency in lavages from patients with and without OC, indicating prevalent somatic evolution in all patients. Driver TP53 mutations, however, presented as significantly larger clones and with higher frequency in lavages from individuals with OC, suggesting that TP53-specific clonal expansions are linked to ovarian cancer development. Our results demonstrate that lavages capture cancer cells, even from early-stage cancers, as well as other clonal expansions and support further exploration of TP53 mutation burden as a potential OC risk factor.

Clonogenic assays improve determination of variant allele frequency of driver mutations in myeloproliferative neoplasms

Molecular diagnostics moves more into focus as technology advances. In patients with myeloproliferative neoplasms (MPN), identification and monitoring of the driver mutations have become an integral part of diagnosis and monitoring of the disease. In some patients, none of the known driver mutations (JAK2V617F, CALR, MPL) is found, and they are termed “triple negative” (TN). Also, whole-blood variant allele frequency (VAF) of driver mutations may not adequately reflect the VAF in the stem cells driving the disease. We reasoned that colony forming unit (CFU) assay–derived clonogenic cells may be better suited than next-generation sequencing (NGS) of whole blood to detect driver mutations in TN patients and to provide a VAF of disease-driving cells. We have included 59 patients carrying the most common driver mutations in the establishment or our model. Interestingly, cloning efficiency correlated with whole blood VAF (p = 0.0048), suggesting that the number of disease-driving cells correlated with VAF. Furthermore, the clonogenic VAF correlated significantly with the NGS VAF (p < 0.0001). This correlation was lost in patients with an NGS VAF <15%. Further analysis showed that in patients with a VAF <15% by NGS, clonogenic VAF was higher than NGS VAF (p = 0.003), suggesting an enrichment of low numbers of disease-driving cells in CFU assays. However, our approach did not enhance the identification of driver mutations in 5 TN patients. A significant correlation of lactate dehydrogenase (LDH) serum levels with both CFU- and NGS-derived VAF was found. Our results demonstrate that enrichment for clonogenic cells can improve the detection of MPN driver mutations in patients with low VAF and that LDH levels correlate with VAF.

Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death.

BackgroundColorectal cancer (CRC) frequently has a dysregulated epigenome causing aberrant up-regulation of oncogenes such as c-MYC. Bromodomain and extra-terminal domain (BET) proteins and histone acetyltransferases (HAT) are epigenetic regulatory proteins that create and maintain epigenetic states supporting oncogenesis. BET inhibitors and HAT inhibitors are currently being investigated as cancer therapeutics due to their ability to suppress cancer-promoting epigenetic modifiers. Due to the extensive molecular crosstalk between BET proteins and HAT proteins, we hypothesized that dual inhibition of BET and HAT could more potently inhibit CRC cells than inhibition of each individual protein.MethodsWe investigated the activity and mechanisms of a dual BET and HAT inhibitor, NEO2734, in CRC cell lines and mouse xenografts. MTS, flow cytometry, and microscopy were used to assess cell viability. qPCR, Western blotting, and immunofluorescent staining were used to assess mechanisms of action.ResultsWe found that NEO2734 more potently suppresses CRC cell growth than first generation BET inhibitors, regardless of the status of common CRC driver mutations. We previously showed that BET inhibitors upregulate DR5 to induce extrinsic apoptosis. In the current study, we show that NEO2734 treatment induces CRC cell apoptosis via both the intrinsic and extrinsic apoptosis pathways. NEO2734 increases p53 expression and subsequently increased expression of the p53-upregulated mediator of apoptosis (PUMA), which is a critical mechanism for activating intrinsic apoptosis. We demonstrate that inhibition of either the intrinsic or extrinsic branches of apoptosis partially rescues CRC cells from NEO2734 treatment, while the dual inhibition of both branches of apoptosis more strongly rescues CRC cells from NEO2734 treatment. Finally, we show that NEO2734 monotherapy is able to suppress tumor growth in CRC xenografts by inducing apoptosis.ConclusionsOur study demonstrates NEO2734 potently suppresses CRC cells in vitro and in vivo by simultaneously upregulating PUMA and DR5 to induce cell death. Further studies of NEO2734 for treating CRC are warranted.