Disease Latency Research Articles

The Role of Quiescence in Acute Myeloid Leukemia Growth

Acute myeloid leukemia (AML) is the most common leukemia in adults and its prognosis is usually poor. Despite the introduction of targeted treatments in recent years, AML remains a high-mortality disease with a 5-year survival of less than 30%. Emerging evidence suggest that therapy failure and leukemia relapse are due to the intra-tumoral genomic and biological heterogeneity. At biological level, AML is hierarchically organized with leukemia stem cells (LSCs) at the apex. LSCs are a rare cell sub-population with self-renewal capacity, responsible for leukemia initiation and maintenance. Standard AML therapies have limited effects on LSCs, mainly due to their property of exiting the cell cycle and enter quiescence. Preliminary data obtained in our group have shown that different leukemia-initiating oncogenes (NPMc+, PML-RARα and MLL-AF9) share the property of enforcing quiescence in pre-leukemic hematopoietic stem cells (HSCs), and that this is critical for the progression and maintenance of the leukemia clone. Underlying molecular mechanisms, however, are unknown. To this end, we have performed an in vivo reverse genetic screening to identify quiescence-related genes indispensable for leukemia growth. Among the identified hits, Socs2, Stat1 and Sytl4 silencing prevented AML outgrowth in vivo. Notably, Socs2 and Stat1 interference increased proliferation, prevented accumulation of quiescent blasts and induced apoptosis. Interestingly, Socs2 and Stat1 silencing exerted no effects on in vitro proliferation, cell cycle distribution or survival of AML blasts, suggesting that their effects are largely dependent on the in vivo leukemia context. Single-cell RNAseq (scRNAseq) analysis of Socs2-interfered blasts showed marked down-regulation of genes characterizing the dormant status of quiescent HSCs, suggesting that loss of quiescence in Socs2-interfered blasts is linked to the loss of their regenerative potential. Since dormancy and self-renewal potential in HSCs are antagonized by prolonged stress signals, we analyzed scRNAseq data for activation of the integrated stress response (ISR). Strikingly, Socs2-interfered cells showed aberrant activation of ATF4, UPR and autophagic transcriptional programs, which was evident in both proliferating and cell cycle restricted blasts, at variance with control blasts that showed increased ISR expression specifically in proliferating cells. Since elevated levels of UPR may act as danger signals and favor immune-mediated clearance in vivo, we analyzed immune-related signaling pathways. Consistently, Socs2-silenced blasts showed a dramatic down-regulation of specific immune check-point molecules, including CD24a, galectin 9 and VISTA, which are involved in the regulation of B cells, T cells, NK cells and macrophages. Based on these findings, we hypothesized that Socs2 regulates the resolution of stressful signals that eventually accumulate in hyper-proliferating AML blasts by allowing cells to enter quiescence and activate pathways of ISR resolution, including upregulation of immune check-point molecules. If blasts fail to enter quiescence, as in in the absence of Socs2 expression, cells maintain sustained ISR activation and downregulate immune check-point molecules, thus triggering immune-mediated cell death. Consistently, the anti-leukemic effect of Socs2-silencing was partially rescued upon propagation into immunocompromised mice. As well, macrophage depletion prolonged disease latency of Socs2-interfered blasts in immunocompetent mice. These findings provide preliminary evidence of the existence, in AML blasts, of an adaptive response to hyperproliferation that involves ISR activation, induction of quiescence and immune evasion. Targeting this adaptive response, as by Socs2 interference, may activate potent mechanisms of AML immune clearance.

In utero origin of myelofibrosis presenting in adult monozygotic twins

The latency between acquisition of an initiating somatic driver mutation by a single-cell and clinical presentation with cancer is largely unknown. We describe a remarkable case of monozygotic twins presenting with CALR mutation-positive myeloproliferative neoplasms (MPNs) (aged 37 and 38 years), with a clinical phenotype of primary myelofibrosis. The CALR mutation was absent in T cells and dermal fibroblasts, confirming somatic acquisition. Whole-genome sequencing lineage tracing revealed a common clonal origin of the CALR-mutant MPN clone, which occurred in utero followed by twin-to-twin transplacental transmission and subsequent similar disease latency. Index sorting and single-colony genotyping revealed phenotypic hematopoietic stem cells (HSCs) as the likely MPN-propagating cell. Furthermore, neonatal blood spot analysis confirmed in utero origin of the JAK2V617F mutation in a patient presenting with polycythemia vera (aged 34 years). These findings provide a unique window into the prolonged evolutionary dynamics of MPNs and fitness advantage exerted by MPN-associated driver mutations in HSCs.

Obesity accelerates acute promyelocytic leukemia in mice and reduces sex differences in latency and penetrance.

ObjectiveObesity has emerged as a prominent risk factor for multiple serious disease states, including a variety of cancers, and is increasingly recognized as a primary contributor to preventable cancer risk. However, few studies of leukemia have been conducted in animal models of obesity. This study sought to characterize the impact of obesity, diet, and sex in a murine model of acute promyelocytic leukemia (APL).MethodsMale and female C57BL/6J.mCG+/PR mice, genetically predisposed to sporadic APL development, and C57BL/6J (wild type) mice were placed on either a high‐fat diet (HFD) or a low‐fat diet (LFD) for up to 500 days.ResultsRelative to LFD‐fed mice, HFD‐fed animals displayed increased disease penetrance and shortened disease latency as indicated by accelerated disease onset. In addition, a diet‐responsive sex difference in APL penetrance and incidence was identified, with LFD‐fed male animals displaying increased penetrance and shortened latency relative to female counterparts. In contrast, both HFD‐fed male and female mice displayed 100% disease penetrance and insignificant differences in disease latency, indicating that the sexual dimorphism was reduced through HFD feeding.ConclusionsObesity and obesogenic diet promote the development of APL in vivo, reducing sexual dimorphisms in disease latency and penetrance.

Characterisation of dust emissions from machined engineered stones to understand the hazard for accelerated silicosis

Engineered stones are novel construction materials associated with a recent upsurge in silicosis cases among workers in the stonemason industry. In order to understand the hazard for the short latency of lung disease among stonemasons, we simulated real-time dust exposure scenario by dry-machining engineered stones in controlled conditions, capturing and analysing the respirable dust generated for physical and chemical characteristics. Natural granite and marble were included for comparison. Cutting engineered stones generated high concentrations of very fine particles (< 1 µm) with > 80% respirable crystalline silica content, in the form of quartz and cristobalite. Engineered stones also contained 8–20% resin and 1–8% by weight metal elements. In comparison, natural stones had far lower respirable crystalline silica (4- 30%) and much higher metal content, 29–37%. Natural stone dust emissions also had a smaller surface area than engineered stone, as well as lower surface charge. This study highlighted the physical and chemical variability within engineered stone types as well as between engineered and natural stones. This information will ultimately help understand the unique hazard posed by engineered stone fabrication work and help guide the development of specific engineering control measures targeting lower exposure to respirable crystalline silica.

Synthetic Matrix Scaffolds Engineer the In Vivo Tumor Immune Microenvironment for Immunotherapy Screening.

Immunotherapy has emerged as one of the most powerful anti-cancer therapies but is stymied by the limits of existing preclinical models with respect to disease latency and reproducibility. Additionally, the influence of differing immune microenvironments within tumors observed clinically and associated with immunotherapeutic resistance cannot be tuned to facilitate drug testing workflows without changing model system or laborious genetic approaches. To address this testing platform gap in the immune oncology drug development pipeline, the authors deploy engineered biomaterials as scaffolds to increase tumor formation rate, decrease disease latency, and diminish variability of immune infiltrates into tumors formed from murine mammary carcinoma cell lines implanted into syngeneic mice. By altering synthetic gel formulations that reshape infiltrating immune cells within the tumor, responsiveness of the same tumor model to varying classes of cancer immunotherapies, including in situ vaccination with a molecular adjuvant and immune checkpoint blockade, diverge. These results demonstrate the significant role the local immune microenvironment plays in immunotherapeutic response. These engineered tumor immune microenvironments therefore improve upon the limitations of current breast tumor models used for immune oncology drug screening to enable immunotherapeutic testing relevant to the variability in tumor immune microenvironments underlying immunotherapeutic resistance seen in human patients.

Evaluation of vestibular-evoked myogenic potential for functional integrity of the brain stem in Kennedy's disease

Objective: To investigate whether vestibular-evoked myogenic potentials(VEMPs) can be used to assess brainstem involvement in patients with Kennedy's disease (KD). Method: This was a case-control study.Twenty consecutive patients with genetically confirmed KD and 20 age-and sex-matched healthy subjects were enrolled from November 2018 to September 2020.All subjects were tested for three types of VEMPs, including cervical VEMP (c-VEMP) recorded by the sternocleidomastoid muscle (parameter:p13, n23), masseter VEMP (m-VEMP) recorded by the masseter muscle(parameter: p11), and ocular VEMP (o-VEMP) recorded by the inferior oblique muscle (parameter n10, p15).The latency of each wave, interside peak latency and interpeak latency of c-VEMP, the corrected amplitude and amplitude asymmetry ratio were recorded. Bilateral sternocleidomastoid muscle (SCMM) electromyography (EMG) was performed. The spinal cord and bulbous muscular atrophy functional rating scale (SBMAFRS) was used for assessment. Results: The mean p13 latency of c-VEMP was (15.5±1.4)ms, which was longer than that of the control group[(13.3±0.9)ms](P<0.05); the mean n23 latency was(25.5±1.4)ms, which was also longer than that of the control group[(22.5±1.0)ms] (P<0.05); the difference of bilateral p13[(2.3±0.6)ms] was significantly higher than that of the control group(P<0.05). The abnormal rates of c-, m-, o-VEMP in KD patients were 75%(15/20), 30%(6/20) and 20%(4/20), respectively. There was a significant positive correlation between c-VEMP latency and course of disease in KD patients(left: r=0.715, 0.695, right: r= 0.708, 0.715, both P<0.05). However, c-VEMP latency was negatively correlated with SBMAFRS score (left: r=-0.701, -0.694, right: r=-0.644, -0.685, both P<0.05). Abnormal rates of SCMM EMG in KD group were as follows: 15%(3/20)of patients showed spontaneous potential in resting state and 45% (9/20) of patients exhibited simple recruitment. Conclusions: The c-VEMP latency is a sensitive tool for detecting lower brainstem involvement in patients with KD, and the degree of damage increases with prolongation of disease course. The o-and m-VEMP abnormalities indicate that some KD patients develop upper brainstem damage.

A class of delay SIQR-V models considering quarantine and vaccination: Validation based on the COVID-19 perspective

To contain the novel SARS-CoV-2 (COVID-19) spreading worldwide, governments generally adopt two measures: quarantining the infected people and vaccinating the susceptible people. To investigate the disease latency's influence on the transmission characteristics of the system, we establish a new SIQR-V (susceptible-infective-quarantined-recovered-vaccinated) dynamic model that focus on the effectiveness of quarantine and vaccination measures in the scale-free network. We use theoretical analysis and numerical simulation to explore the evolution trend of different nodes and factors influencing the system stability. The study shows that both the complexity of the network and latency delay can affect the evolution trend of the infected nodes in the system. Still, only latency delay can destroy the stability of the system. In addition, through the parameter sensitivity analysis of the basic reproduction number, we find that the effect of the vaccination parameter α on the basic reproduction number R0 is more significant than that of transmission rate β and quarantine parameter σ. It shows that vaccination is one of the most effective public policies to prevent infectious diseases’ spread. Finally, we calculate the basic reproduction numbers that are greater than one for Germany and Pakistan under COVID-19 and validate the model’s effectiveness based on the disease data of COVID-19 in Germany. The results show that the changing trend of the infected population in Germany based on the SIQR-V model is roughly the same as that reflected by the actual epidemic data in Germany. Therefore, providing suggestions and guidance for treating infectious diseases based on this model can effectively reduce the harm caused by the outbreak of contagious diseases.

The Molecular Landscape of KMT2A-Rearranged Leukemia from Infancy to Adulthood Reveals Age and Leukemia-Specific Mutational Patterns

Genetic rearrangements involving the KMT2A gene (KMT2A-R) are seen in around 10% of acute leukemia overall. KMT2A-R occurs in all ages and usually correlates with high-risk clinical features, in particular in infants aged 0-12 months of age with acute lymphoblastic leukemia (ALL). To uncover age- and leukemia-subtype specific molecular patterns in KMT2A-R ALL and acute myeloid leukemia (AML), we performed whole genome (WGS), whole exome (WES), and RNA-sequencing on a well-annotated Nordic KMT2A-R cohort of 104 patients, including infant ALL (n=33), childhood ALL (n=18), adult ALL (n=15), and pediatric AML (n=38) patients. For 77 patients, we performed WGS (40x) at diagnosis and remission as well as WES (140x) on the diagnostic sample, and remaining patients underwent WES only (n=27). RNA-sequencing was performed on 58 cases with available RNA.Twenty-two genes were recurrently altered and remarkably, NRAS, KRAS, FLT3, PAX5, TP53, CDKN2A/B and IKZF1 accounted for 70% of mutations. The landscape of mutations suggested the presence of leukemia and age-specific associations with MYST4, PTPN11, and SETD2 uniquely altered in AML and PIK3CD, DNAH11, NOTCH1, CSMD3 and CDKN2A/B in ALL. Some genes were mutated in both KMT2A-R ALL and AML, but were more common in one disease, such as FLT3 and KRAS in AML and PAX5, TP53 and IKZF1 in ALL. Moreover, age-associated patterns were seen in ALL with NRAS more frequently mutated than KRAS in infant ALL (26% vs 15%), and KRAS more frequently mutated than NRAS in childhood ALL (24% vs 18%), with adult ALL having fewer such mutations (NRAS 13%; KRAS 7%). Alterations of CDKN2A/B and TP53 were absent in infant ALL, detected in childhood and adult ALL only. PAX5 alterations were primarily detected in childhood ALL (22%, 9% infant ALL, 7% adult ALL), with all three PAX5-altered infant cases having the KMT2A-MLLT3 fusion gene. Finally, KMT2A-R pediatric AML had the highest fraction of FLT3 mutations (24%, 9% infant ALL, 11% childhood ALL, 0% adult ALL) and all but one mutation occurred in KMT2A-MLLT3 rearranged cases and most were kinase domain point mutations.We next expanded our analysis to include non-recurrent alterations. PI3K/RAS pathway alterations were detected across ages and subtypes with the highest fraction in pediatric AML (63%) and the lowest in adult ALL (27%, 43% infant ALL, 41% childhood ALL). Further, cell cycle related genes were primarily mutated in childhood (39%) and adult ALL cases (33%) and rarer in infant ALL (12%) and pediatric AML (16%) and genes within the B-cell pathway were more commonly altered in childhood ALL (29%) than in infant ALL (9%). Finally, in line with our previous study (Andersson et al, Nat Genet 2015) epigenetic mutations were absent in infant ALL, but present in 20-35% of the other patients.RNA-sequencing identified the KMT2A-fusion in 56/58 cases, with low exonic coverage preventing detection of the fusion in two cases. The reciprocal KMT2A fusion was only expressed in 13/39 cases where it was predicted to be expressed based on karyotype or whole genome sequencing data with 11/13 cases having the KMT2A-AFF1 fusion gene. In addition, RNA-sequencing identified 6 in-frame and 12 out-of-frame fusion genes that had formed either as part of the KMT2A-R itself or that were independent genetic events. Further, a novel in-frame KMT2A-ACIN1 fusion was identified in a child aged 1 year with B-precursor ALL. ACIN1 encodes Apoptotic Chromatin Condensation Inducer 1 and the fusion was formed through an insertion of 14q11 into 11q23. ACIN1 is also rearranged as part of the ACIN1-NUTM1 that we identified in an infant with ins(15;14)(q22;q11.2q32.1) (Andersson et al Nat Genet 2015). To study the ability of KMT2A-ACIN1 to induce leukemia in mice, we injected retrovirally transduced mouse bone marrow cells containing the fusion into syngeneic mice and KMT2A-MLLT3 was used as control. All mice succumbed to disease at an average of 112 days for KMT2A-ACIN1 (n=12) and 63 days for KMT2A-MLLT3 (n=5) and mice displayed splenomegaly and leukocytosis with an immunophenotype indicative of AML. Primary leukemia cells isolated from moribund mice gave rise to leukemia in sublethally irradiated recipients with reduced disease latency. In conclusion, these results highlight the differential molecular patterns in KMT2A-R leukemia across infancy to adulthood thereby providing novel pathogenetic insight. DisclosuresNo relevant conflicts of interest to declare.

Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML.We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints.Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P<0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. [Display omitted] DisclosuresGopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

The Krüppel-like Factor 4 Sustains a Leukemic Gene Expression Profile in MLL-AF9-Driven Acute Myeloid Leukemia

Among hematological malignancies, acute myeloid leukemia (AML) confers poor prognosis and limited progress has been made in the translation of decades of research into improved clinical outcomes. The current paradigm is that eradication of leukemia stem cells (LSCs) represents an avenue for overcoming relapse and refractory disease, but therapy focusing on eradicating this leukemic population has not been developed to-date. Further studies of unique signaling pathways and vulnerabilities in LSCs are warranted to design targeted therapies that could impact patient outcomes. To evaluate whether the stemness transcription factor Krüppel-like Factor 4 (KLF4) is important in the progression of AML, we retrovirally transduced MLL-AF9 into Klf4 fl/fl(fl/fl)and Klf4 fl/flVav-Cre (Δ/Δ) lineage − Sca-1 + c-Kit + (LSK) bone marrow cells and transplanted into C57BL/6 recipients. Here we report that the KLF4 promotes disease progression in the MLL-AF9-driven syngeneic AML mouse model. Strikingly, Δ/Δ AMLs exhibited improved disease latency and penetrance, and a seven-fold reduction in leukemia-initiating cell frequency in a secondary transplantation study. Δ/Δ LSCs, defined as leukemic granulocyte macrophage progenitors (L-GMP), demonstrated lessened clonogenicity in methylcellulose cultures and reduced representation of cells in the G 2/M phase of the cell cycle. RNAseq analysis of L-GMP revealed decreased expression of hematopoietic and leukemic stemness gene sets such as RAS signaling, and induction of inflammatory response gene (TNF-α, IFNα, IFNβ) pathways in Δ/Δ LSCs. To evaluate human relevance, we used CRISPR-Cas9 based targeted deletion of the human KLF4 gene in a MLL-AF9 PDX line and observed improved survival and defects in expansion as seen in the syngeneic mouse model . Lastly, to correlate KLF4-associated signaling present in murine AML LSCs with human AML, we used CRISPR-Cas9-based targeted deletion of KLF4 in MOLM-13 (KO) to generate two validated clones. MOLM-13 KO cells showed reduced cell proliferation in vitro and in vivo. Further, RPPA analysis revealed reduced RAS pathway activity (IR-β, β-Raf), accumulation of proteins associated with the S and G 1 phases (e.g., CDKN2A, p21, Histone H3, CENP-A), and decrease expression in regulators of the G 2/M checkpoint (e.g., Aurora A, B, Chk1, Plk1, Wee1, Cyclin B, pCDK1). Collectively, our data suggest a mechanism in which KLF4 contributes to AML disease by establishing a gene expression profile supporting stemness of AML LSCs. DisclosuresNo relevant conflicts of interest to declare.

Predictors of relapse in MOG antibody associated disease: a cohort study

ObjectiveTo identify factors predictive of relapse risk and disability in myelin oligodendrocyte glycoprotein associated disease (MOGAD).SettingPatients were seen by the neuromyelitis optica spectrum disorders (NMOSD) service in Liverpool, UK, a...

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

Liver injury caused by fenofibrate within 48\xa0h after first administration: a case report

BackgroundFenofibrate is commonly used in the treatment of dyslipidemia. Fenofibrate is related to mild aminotransferase elevations and in some cases severe chronic injury such as fibrosis or cirrhosis, resulting in liver transplantation or death. The latency of disease has been reported to range between weeks to years.Case presentationA 63 years old male with hypertriglyceridemia developed symptoms of fatigue and anorexia 48 h after taking fenofibrate for the first time. The patient’s aminotransferase level was more than 10 times ULN. Immediately, fenofibrate was discontinued and aminotransferase level returned to normal 23 days later. To assess causality between the drug and liver damage, the standardized Roussel Uclaf Causality Assessment Method (RUCAM) was used. The patient's RUCAM score was 7, which fell in the group of “probable”. Eight months later, follow-up examination suggested the liver function was normal.ConclusionsWeakness, fatigue and abnormal liver function during fenofibrate therapy should be closely monitored and trigger prompt withdrawal if these symptoms occur.

Abstract 2884: Wnt11 dictates the patterns and latency of pulmonary micrometastatic disease in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a systemic disease characterized by early metastasis involving numerous pro-metastatic pathways. However, dominant drivers of early PDAC cell dissemination and metastasis remain poorly defined. The non-canonical Wnt-PCP pathway has been shown to promote cancer cell motility predominantly through Wnt ligand-mediated activation of frizzled receptors that result in cytoskeleton rearrangements. As one of these Wnt ligands, Wnt11, has been previously implicated in metastasis, we tested if Wnt11 significantly affects PDAC metastasis in vivo. Methods: LSL-KrasG12D/+, p53wmR172H/+, and p48-Cre alleles were crossed with and without a conditional Wnt11fl allele to generate cohorts of Wnt11 wildtype and knockout mice in the well-established KPC PDAC mouse model (KPwmC, n = 22; KPwmC-Wnt11fl/fl, n = 37). Both cohorts included mice with a conditional LSL-ROSA26-tdTomato reporter to identify and enumerate distant metastases. Mice were sacrificed when euthanasia criteria were met, and metastases were visualized via full organ fluorescence microscopy to identify tdTomato(+) signals. Liver and lung metastases were enumerated and measured through automated color range selection and manual counting. Metastases were classified by diameter as micrometastases (&amp;lt;100 uM), intermediate metastases (100-499 uM), or macrometastases (≥500 uM). Results: Primary pancreatic tumors from KPwmC and KPwmC-Wnt11fl/fl mice were similar in histologic appearance without gross alterations in peritumoral stroma. There were no differences in median overall survival between KPwmC and KPwmC-Wnt11fl/fl mice (185 vs. 193 days, p = 0.92). Pulmonary micrometastatic disease in mice &amp;lt;170 days at euthanasia was significantly lower in KPwmC-Wnt11fl/fl mice relative to KPwmC mice (mean number of metastases 88.8 vs. 1780, respectively; p = 0.009). Moreover, while pulmonary metastases in KPwmC mice were diffuse and miliary, the lungs of KPwmC-Wnt11fl/fl mice contained only scant, scattered metastases. No significant differences were observed in the mean number of intermediate-sized (0.5 vs. 27.4, p = 0.17) or macrometastases (0.0 vs 1.6, p = 0.13). Differences in metastatic tumor size or burden were not observed in liver metastases between each cohort. Conclusions: Wnt11 affects the pattern and latency of early metastasis to the lung but does not affect any stage of hepatic metastasis. The role of Wnt11 in PDAC metastasis is context dependent and may be related to factors inherent in the pre-metastatic niche. Citation Format: Eileen C. Donovan, Bingbing Dai, Jenying Deng, Christian M. Siangco, Xinqun Li, Erika Y. Faraoni, Sylvia M. Evans, Michael P. Kim. Wnt11 dictates the patterns and latency of pulmonary micrometastatic disease in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2884.

Abstract 2584: Leukemia latency in the mCG-PML-RARα mouse model is impacted by sex and obesity

Abstract Human leukemias have been reported to show sexual dimorphism in both incidence and behavior. However, few studies of this observation have been conducted in animal models. We used the C57BL/6J mouse, genetically engineered to contain the PML-RARA fusion gene at the mouse cathepsin G (mCG) locus, as a model (B6.mCG-PML-RARα) of acute promyelocytic leukemia (APL) to interrogate the impact of sex and diet variation on disease latency as measured by leukemia-free survival. At 30 days of age, control (WT) and mutant (mCG+/PR) mice were separated by sex and provided with high fat (HF) [57% coconut oil fat] or low fat (LF) [10% coconut oil fat] diets. Mice were followed with serial blood collections and sacrificed when peripheral blood revealed signs of leukemia development including elevated granulocytes (WBC &amp;gt;20 K/μL), anemia (Hb &amp;lt;11 g/dL), and thrombocytopenia (PLT &amp;lt;600 K/μL). At necropsy, leukemic mice were noted to have marked splenomegaly (0.73±0.40 g) compared to controls (0.30±0.19 g). However, splenomegaly was not affected by diet in WT mice, as spleen size was consistent in both HF and LF diet-fed animals. mCG+/PR male mice fed HF diets showed greater weight gain and were significantly (P&amp;lt;0.001) heavier than mCG+/PR male mice fed LF diets (45.1±6.6 g versus 31.3±2.9 g at 150 days on diet). Female mCG+/PR mice fed HF diets showed greater weight gain than female mCG+/PR mice fed LF diets (31.8±5.5 g versus 23.2±1.9 g at 150 days on diet). In addition, mCG+/PR male mice fed HF diets were significantly heavier than female mCG+/PR mice fed HF diets (45.1±6.6 g versus 31.8±5.5 g at 150 days on diet). This sexual dimorphism in weight accumulation has been previously noted and attributed in part to ovarian function. The longest latency for the appearance of APL was shown in female mCG+/PR mice fed LF diets, where a median leukemia-free survival (MLFS) of &amp;gt;425 days was observed. In contrast, male mCG+/PR mice fed LF diets showed a reduced MLFS of 360 days of age, indicating a difference in latency of 65 days, thereby demonstrating sexual dimorphism in the appearance of APL. HF diet accelerated the appearance of APL in both female (MLFS = 277 days of age) and male mCG+/PR mice (MLFS = 277 days of age), which also displayed a difference in latency of 65 days. Thus, HF diet accelerated appearance of APL by 148 days in male and 148 days in female mCG+/PR mice. There were no signs of leukemia development in WT mice on either HF or LF diets. There was no splenomegaly observed in WT mice on either HF or LF diets. Thus, although HF diet and obesity accelerate the onset of APL in B6.mCG-PML-RARα mice, sexual dimorphism remains, with females showing extended latency compared to males under conditions of both HF and LF diets. Overall, our studies provide a robust mouse model to study the mechanisms by which sex impacts leukemia latency, as well as demonstrate that obesity accelerates leukemia development without affecting sexual dimorphism. Citation Format: John W. Kincaid, Annie E. Hill-Baskin, Nathan A. Berger. Leukemia latency in the mCG-PML-RARα mouse model is impacted by sex and obesity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2584.

FLT3N676K drives Myeloid Leukemia in a Xenograft Model of KMT2A-MLLT3 leukemogenesis

Geneticrearrangements of the KMT2A gene (previously MLL) are found in approximately 10% of human acute leukemia. However, in acute lymphoblastic leukemia (ALL) occurring in infants less than 1 year of age, a leukemia subtype with a particularly poor prognosis, close to 80% have a KMT2A -rearrangement (KMT2A -R). The KMT2A -Rs display an unusual lineage promiscuity and even though the specific fusion gene and cell-of-origin likely has an impact, the mechanisms influencing lineage determination in KMT2A -leukemogenesis remains largely unknown. Recently, when defining the landscape of mutations in infant KMT2A -R ALL, we identified a novel FLT3N676K mutation in the tyrosine kinase domain that caused factor independent growth of the interleukin-3 dependent leukemia cell line Ba/F3 (Andersson et al., 2015). Notably, FLT3N676K was the most common FLT3 mutation in our cohort which also included non-infant KMT2A -R ALL and acute myeloid leukemia (AML).Herein, we studied the biological impact of the FLT3N676K mutationin KMT2A-MLLT3 driven leukemogenesis in human hematopoietic cells. Retroviral co-expression of FLT3N676K and KMT2A-MLLT3 in human CD34+ enriched cord blood cells transplanted to NOD.Cg- PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice primarily caused AML and rarely ALL. By contrast, expression of KMT2A-MLLT3 alone caused ALL, dual-phenotype leukemia (DPL) or bilineal leukemia (BLL), and rarely AML. Mice receiving cells expressing KMT2A-MLLT3 alone(n=23) or KMT2A-MLLT3+FLT3N676K (n=26) succumbed to disease at a median latency of 119 and 107.5 days respectively, with no significant difference in disease latency between the groups. However, examining all KMT2A-MLLT3 leukemias combined, with and without FLT3N676K, highlighted that AML developed with a significantly shorter latency as compared ALL and DPL. Analysis of the bone marrow of moribund mice revealed that almost all cells (98.3±1.9%) expressed human CD45 (hCD45+). Mice receiving FLT3N676K alone were sacrificed at day 209 or 223 with no sign of disease.Gene expression analysis of highly purified leukemia cells revealed that KMT2A-MLLT3 + FLT3N676K enforced Myc- and Myb transcriptional modules and increased expression of negative feedback regulators of the RAS signaling pathway. Further, KMT2A-MLLT3 DPL and ALL cells had highly similar gene expression profiles with high expression of PAX5, IKZF1/2, and EBF1, indicating that the DPL cells are lymphoid-primed and aberrantly express CD33. Both KMT2A-MLLT3 DPL and ALL cells displayed weak but distinct expression of CEBPA suggesting that KMT2A-MLLT3 DPL cells originate from a progenitor cell that express CEBPA . Consistent with the high similarities of DPL and ALL cells, a fraction of DPL cells became CD19+CD33-ex vivo in lymphoid culture conditions. Further, we identified enrichment of SMAD signaling in KMT2A-MLLT3 lymphoid leukemia cells as well as in primary human KMT2A -R ALL, suggesting that TGFB-regulation is different in lymphoid and myeloid KMT2A -driven leukemia. Consistent with this, human KMT2A -R ALL cell lines were more sensitive towards a specific inhibitor of SMAD3 as compared to KMT2A -R AML cell lines, suggesting that inhibition of SMAD signaling may be a fruitful approach for KMT2A -R ALL.Taken together, herein we show that co-expression of KMT2A-MLLT3 and FLT3N676K in human cord blood cells preferentially drive expansion of acute myeloid leukemia cells. The data suggest that a cooperating lesion can influence the immunophenotype of the resultant leukemia, at least in a xenograft setting, thereby adding new insight into the biology of KMT2A -R leukemia and the mechanism by which leukemia-associated genetic lesions cooperate in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

H3K27M/I Mutations Promote Context-Dependent Transformation in Acute Myeloid Leukemia with RUNX1 Alterations

Neomorphic missense mutations in histone H3 genes (most notably K27M and K36M) are frequent in solid cancers such as diffuse intrinsic pontine gliomas (DIPG), supratentorial glioblastoma multiforme (GBM) or chondroblastomas. Despite high incidences of these mutations and their profound impact on global histone H3 lysine methylation, their relevance has not been studied in hematologic cancers.Here, we report the first identification of histone H3 mutations in AML. We find that two of 415 AML patients in the Leucegene cohort and one of 200 patients in the TCGA cohort carry either K27M (n=2) or K27I (n=1) mutations in one of twelve canonical H3 genes. No K36M or other lysine-to-methionine mutations on any of the H3 genes, and no K27M mutation on any of the replication-independent H3.3 variant genes were detected in the same cohorts. H3K27 mutations invariably occurred in the context of either truncating RUNX1 mutations or t(8;21)(q22;q22), suggesting an association with altered RUNX1 function (p < 0.003; Fisher exact test). Characterization of two of the identified H3K27 mutant patient specimens revealed that both samples contained major sub-clones with markedly reduced H3K27me2/3 levels. The H3K27 mutants but not co-occurring mutant ASXL2 or USP7 alleles were only detectable in the H3K27me2/3-low sub-clones, demonstrating that the H3K27 mutations had the most profound impact on H3K27me2/3 in these patients. Moreover, although a heterozygous TET2S1203R mutation was equally present in both H3K27me2/3-high and low sub-fractions, a homozygous RUNX1L98Sfs*22 mutation was restricted to the H3K27me2/3-low sub-clone, further strengthening the possibility that impaired RUNX1 function and reduced H3K27me2/3 associate with each other.We tested the possibility that H3K27 mutants may specifically collaborate with the product of the t(8;21) translocation (AML1-ETO-9a ; AE9a) in transduced mouse hematopoietic stem and progenitor cells (HSPCs). Selectivity of this collaboration was assessed using the combination of the H3K27 mutant gene with the MLL-AF9 fusion product. Whereas expression of H3 WT resulted in a mild competitive proliferation disadvantage in both AE9a and MLL-AF9 cells, expression of H3.3K27I/M conferred a proliferative advantage in AE9a cells in three of four H3K27I -infected and in all H3K27M- infected cultures. This was not observed in MLL-AF9 cells, where H3K27I/M expressing cells were progressively outcompeted in the same culture period. H3K27I/M -infected AE9a cells exhibited significant reductions in H3K27me2/3 and elevated levels of H3K27 acetylation (H3K27ac) compared to H3WT -infected control cells despite undiminished Ezh1, Ezh2 and Suz12 protein levels. In the context of AE9a driven AML in vivo, expression of H3K27M caused a striking disease acceleration leading to all mice reaching end stage disease with a median of 88 days, in contrast to recipients of AE9a ; empty vector or AE9a ; H3WT cells, of which none or only two of nine transplanted mice succumbed to AML within 330 days, respectively (p<0.0001, Log-rank test). Seven of nine AE9a ; H3.3K27I transplanted mice developed AML with a median latency of 208 days (p=0.0004, Log-rank test, compared to controls). Importantly, combined expression of H3K27I or H3K27M with MLL-AF9 did not change disease latencies compared to control groups, indicating selectivity.We next tested the influence of the H3K27M mutation on the differentiation and proliferation of human cord blood CD34+ HSPCs ex vivo . Relative to controls, H3.3K27M expression led to an 8-fold net expansion of a phenotypically primitive CD34+/CD45RA-/CD90+ cell population, which surprisingly lacked expression of the CD133 stem cell marker. Interestingly, granulocytic/granulo-monocytic colony-forming cells (CFU-G/GM) but not multipotent colony-forming cells (granulo/erythroid/monocytic/megakaryocytic; CFU-GEMM) were expanded in 14d cultures initiated with H3K27M transduced CD34+ CB cells when compared to controls. Xenografts of transduced CD34+ CB cells indicated a marginal loss in in vivo reconstitution activity by K27M-transduced cells, mostly at 6 and 12-week time-points.Taken together, our results represent the first description of neomorphic H3K27M/I mutations in AML. We demonstrate that these mutations have a profound impact on H3K27me2/3 and provide evidence that they selectively collaborate with impaired RUNX1 function. DisclosuresNo relevant conflicts of interest to declare.

Label-Free Quantitative Proteomic Analysis of Three Strains of Viscerotropic Leishmania Isolated from Patients with Different Epidemiological Types of Visceral Leishmaniasis in China.

There are three epidemiological types of visceral leishmaniasis in China, which are caused by Leishmania strains belonging to the L. donovani complex. The mechanisms underlying their differences in the population affected, disease latency, and animal host, etc., remain unclear. We investigated the protein abundance differences among Leishmania strains isolated from three types of visceral leishmaniasis endemic areas in China. Promastigotes of the three Leishmania strains were cultured to the log phase and harvested. The protein tryptic digests were analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free quantitative analysis. The MS experiment was performed on a Q Exactive mass spectrometer. Raw spectra were quantitatively analyzed with the MaxQuant software (ver 1.3.0.5) and matched with the reference database. Differentially expressed proteins were analyzed using the bioinformatics method. The MS analysis wasrepeatedthreetimes for each sample. A total of 5012 proteins were identified across the KS-2, JIASHI-5 and SC6 strains in at least 2 of the three samples replicate. Of them, 1758 were identified to be differentially expressed at least between 2 strains, including 349 with known names. These differentially expressed proteins with known names are involved in biological functions such as energy and lipid metabolic process, nucleotide acid metabolic process, amino acid metabolic process, response to stress, cell membrane/cytoskeleton, cell cycle and proliferation, biological adhesion and proteolysis, localization and transport, regulation of the biological process, and signal transduction. The differentially expressed proteins and their related biological functions may shed light on the pathogenicity of Leishmania and targets for the development of vaccines and medicines.

Palmitoylethanolamide: A Natural Compound for Health Management.

All nations which have undergone a nutrition transition have experienced increased frequency and falling latency of chronic degenerative diseases, which are largely driven by chronic inflammatory stress. Dietary supplementation is a valid strategy to reduce the risk and severity of such disorders. Palmitoylethanolamide (PEA) is an endocannabinoid-like lipid mediator with extensively documented anti-inflammatory, analgesic, antimicrobial, immunomodulatory and neuroprotective effects. It is well tolerated and devoid of side effects in animals and humans. PEA’s actions on multiple molecular targets while modulating multiple inflammatory mediators provide therapeutic benefits in many applications, including immunity, brain health, allergy, pain modulation, joint health, sleep and recovery. PEA’s poor oral bioavailability, a major obstacle in early research, has been overcome by advanced delivery systems now licensed as food supplements. This review summarizes the functionality of PEA, supporting its use as an important dietary supplement for lifestyle management.

Downregulation of MTSS1 in acute myeloid leukemia is associated with a poor prognosis, chemotherapy resistance, and disease aggressiveness

Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML.