Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing.

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.

Unusually High Prevalence of Stroke and Cerebral Vasculopathy in Hemoglobin SC Disease: A Retrospective Single Institution Study.

INTRODUCTION: Unlike homozygous hemoglobin SS (HbSS) disease, stroke is a rare complication in hemoglobin SC (HbSC) disease. However, recent studies have demonstrated a high prevalence of silent stroke in HbSC disease. The factors associated with stroke and cerebral vasculopathy in the HbSC population are unknown. METHODS: We conducted a retrospective study of all patients with sickle cell disease treated at the University of Missouri, Columbia, over an 18-year period (2000-2018). The goal of the study was to characterize the silent, overt stroke, and cerebral vasculopathy in HbSC patients and compare them to patients with HbSS and HbS/ß thalassemia1 (thal) in this cohort. We also analyzed the laboratory and clinical factors associated with stroke and cerebral vasculopathy in the HbSC population. RESULTS: Of the 34 HbSC individuals, we found that the overall prevalence of stroke and cerebral vasculopathy was 17.7%. Only females had evidence of stroke or cerebral vasculopathy in our HbSC cohort (33.3%, p = 0.019). Time-averaged means of maximum velocities were lower in the HbSC group than the HbSS group and did not correlate with stroke outcome. Among HbSC individuals, those with stroke and cerebral vasculopathy had a marginally higher serum creatinine than those without these complications (0.77 mg/dL vs. 0.88 mg/dL, p = 0.08). Stroke outcome was associated with recurrent vaso-occlusive pain crises (Rec VOCs) (75 vs. 25%, p = 0.003) in HbSC patients. The predominant cerebrovascular lesions in HbSC included microhemorrhages and leukoencephalopathy. CONCLUSION: There is a distinct subset of individuals with HbSC who developed overt, silent stroke, and cerebral vasculopathy. A female predominance and association with Rec VOCs were identified in our cohort; however, larger clinical trials are needed to identify and confirm specific clinical and laboratory markers associated with stroke and vasculopathy in HbSC disease.

L-leucine improves anemia and growth in patients with transfusion-dependent Diamond-Blackfan anemia: Results from a multicenter pilot phase I/II study from the Diamond-Blackfan Anemia Registry.

BACKGROUND: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. PROCEDURE: Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). RESULTS: This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. CONCLUSIONS: L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

An in vivo multiplexed small-molecule screening platform.

Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.

ZRSR2 mutation in a child with refractory macrocytic anemia and Down Syndrome.

Here we report a case of refractory macrocytic anemia with a spliceosomal point mutation involving the ZRSR2 gene in a child with Down syndrome (DS). Such mutations have been shown to cause refractory macrocytic anemia and myelodysplastic syndrome (MDS) in elderly individuals. We report the hematological indices of a child with DS and a ZRSR2 spliceosomal mutation. DS is known to produce macrocytic anemia but does not lead to transfusion dependence. In this case, the ZRSR2 mutation was the likely implicating factor for severe transfusion-dependent anemia in a child with DS. The clinical implication of a ZRSR2 mutation in a child with DS has not been previously described and warrants close surveillance to detect potential insidious transformation to MDS.

Predissociation of high-lying Rydberg states of molecular iodine via ion-pair states.

Velocity map imaging of the photofragments arising from two-photon photoexcitation of molecular iodine in the energy range 73 500-74 500 cm(-1) covering the bands of high-lying gerade Rydberg states [(2)Π1/2]c6d;0g (+) and [(2)Π1/2]c6d;2g has been applied. The ion signal was dominated by the atomic fragment ion I(+). Up to 5 dissociation channels yielding I(+) ions with different kinetic energies were observed when the I2 molecule was excited within discrete peaks of Rydberg states and their satellites in this region. One of these channels gives rise to images of I(+) and I(-) ions with equal kinetic energy indicating predissociation of I2 via ion-pair states. The contribution of this channel was up to about 50% of the total I(+) signal. The four other channels correspond to predissociation via lower lying Rydberg states giving rise to excited iodine atoms providing I(+) ions by subsequent one-photon ionization by the same laser pulse. The ratio of these channels varied from peak to peak in the spectrum but their total ionic signal was always much higher than the signal of (2 + 1) resonance enhanced multi-photon ionization of I2, which was previously considered to be the origin of ionic signal in this spectral range. The first-tier E0g (+) and D(')2g ion-pair states are concluded to be responsible for predissociation of Rydberg states [(2)Π1/2]c6d;0g (+) and [(2)Π1/2]c6d;2g, respectively. Further predissociation of these ion-pair states via lower lying Rydberg states gives rise to excited I(5s(2)5p(4)6s(1)) atoms responsible for major part of ion signal. The isotropic angular distribution of the photofragment recoil directions observed for all channels indicates that the studied Rydberg states are long-lived compared with the rotational period of the I2 molecule.

KDM5B predicts temozolomide-resistant subclones in glioblastoma.

Adaptive plasticity to the standard chemotherapeutic temozolomide (TMZ) leads to glioblastoma progression. Here, we examine early stages of this process in patient-derived cellular models, exposing the human lysine-specific demethylase 5B (KDM5B) as a prospective indicator for subclonal expansion. By integration of a reporter, we show its preferential activity in rare, stem-like ALDH1A1+ cells, immediately increasing expression upon TMZ exposure. Naive, genetically unmodified KDM5Bhigh cells phosphorylate AKT (pAKT) and act as slow-cycling persisters under TMZ. Knockdown of KDM5B reverses pAKT levels, simultaneously increasing PTEN expression and TMZ sensitivity. Pharmacological inhibition of PTEN rescues the effect. Interference with KDM5B subsequent to TMZ decreases cellular vitality, and clonal tracing with DNA barcoding demonstrates high individual levels of KDM5B to predict subclonal expansion already before TMZ exposure. Thus, KDM5Bhigh treatment-naive cells preferentially contribute to the dynamics of drug resistance under TMZ. These findings may serve as a cornerstone for future biomarker-assisted clinical trials.

A Lung Cancer Mouse Model Database.

Lung cancer, the leading cause of cancer mortality, exhibits diverse histological subtypes and genetic complexities. Numerous preclinical mouse models have been developed to study lung cancer, but data from these models are disparate, siloed, and difficult to compare in a centralized fashion. Here we established the Lung Cancer Mouse Model Database (LCMMDB), an extensive repository of 1,354 samples from 77 transcriptomic datasets covering 974 samples from genetically engineered mouse models (GEMMs), 368 samples from carcinogen-induced models, and 12 samples from a spontaneous model. Meticulous curation and collaboration with data depositors have produced a robust and comprehensive database, enhancing the fidelity of the genetic landscape it depicts. The LCMMDB aligns 859 tumors from GEMMs with human lung cancer mutations, enabling comparative analysis and revealing a pressing need to broaden the diversity of genetic aberrations modeled in GEMMs. Accompanying this resource, we developed a web application that offers researchers intuitive tools for in-depth gene expression analysis. With standardized reprocessing of gene expression data, the LCMMDB serves as a powerful platform for cross-study comparison and lays the groundwork for future research, aiming to bridge the gap between mouse models and human lung cancer for improved translational relevance.

A Sequential Targeting Strategy Interrupts AKT-Driven Subclone-Mediated Progression in Glioblastoma.

PURPOSE: Therapy resistance and fatal disease progression in glioblastoma are thought to result from the dynamics of intra-tumor heterogeneity. This study aimed at identifying and molecularly targeting tumor cells that can survive, adapt, and subclonally expand under primary therapy. EXPERIMENTAL DESIGN: To identify candidate markers and to experimentally access dynamics of subclonal progression in glioblastoma, we established a discovery cohort of paired vital cell samples obtained before and after primary therapy. We further used two independent validation cohorts of paired clinical tissues to test our findings. Follow-up preclinical treatment strategies were evaluated in patient-derived xenografts. RESULTS: We describe, in clinical samples, an archetype of rare ALDH1A1+ tumor cells that enrich and acquire AKT-mediated drug resistance in response to standard-of-care temozolomide (TMZ). Importantly, we observe that drug resistance of ALDH1A1+ cells is not intrinsic, but rather an adaptive mechanism emerging exclusively after TMZ treatment. In patient cells and xenograft models of disease, we recapitulate the enrichment of ALDH1A1+ cells under the influence of TMZ. We demonstrate that their subclonal progression is AKT-driven and can be interfered with by well-timed sequential rather than simultaneous antitumor combination strategy. CONCLUSIONS: Drug-resistant ALDH1A1+/pAKT+ subclones accumulate in patient tissues upon adaptation to TMZ therapy. These subclones may therefore represent a dynamic target in glioblastoma. Our study proposes the combination of TMZ and AKT inhibitors in a sequential treatment schedule as a rationale for future clinical investigation.

Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

An inducible Tet-Off-H2B-GFP lentiviral reporter vector for detection and in vivo isolation of label-retaining cells.

Many regenerative cells are label-retaining cells (LRCs) due to their ability to keep a DNA label over a prolonged time. Until recently, isolation of vital LRCs was hampered due to the necessary use of fixation methods. To circumvent this, we generated a lentiviral-(HIV-1) based vector expressing a Tet-Off controlled histone 2B-GFP (Tet-Off-H2B-GFP) reporter gene for the detection and isolation of viable LRCs. In initial experiments, the vector was successfully used to infect 2- and 3-dimensional tissue culture models. Infected cultures from skin and pancreatic cells showed a very tight regulation of H2B-GFP, were sensitive to minimal amounts of doxycycline (Dox) and had a stable transgenic expression over the time of this study. Our lentiviral vector represents a reliable and easy to handle system for the successful infection, detection and isolation of LRCs from various tissues in vitro, in vivo and ex vivo.

Barcoding Technology for Multiplexed Analysis of Metastatic Ability In Vivo.

DNA barcoding allows the quantitative, biomarker-free tracking of individual cell populations in mixed/heterogeneous cell pools. Here, we describe a multiplexed in vivo screening platform based on DNA barcoding technology to interrogate compound libraries for their effect on metastatic seeding in vivo. We apply next-generation sequencing (NGS) technology to quantitatively analyze high-throughput compound screening in mice. Up to 96 compounds and controls can be screened for their effect on metastatic ability in a single mouse.

Unraveling Tumor Heterogeneity by Using DNA Barcoding Technologies to Develop Personalized Treatment Strategies in Advanced-Stage PDAC.

Tumor heterogeneity is a hallmark of many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), and an inherent consequence of the clonal evolution of cancers. As such, it is considered the underlying concept of many characteristics of the disease, including the ability to metastasize, adapt to different microenvironments, and to develop therapy resistance. Undoubtedly, the high mortality of PDAC can be attributed to a high extent to these properties. Despite its apparent importance, studying tumor heterogeneity has been a challenging task, mainly due to its complexity and lack of appropriate methods. However, in recent years molecular DNA barcoding has emerged as a sophisticated tool that allows mapping of individual cells or subpopulations in a cell pool to study heterogeneity and thus devise new personalized treatment strategies. In this review, we provide an overview of genetic and non-genetic inter- and intra-tumor heterogeneity and its impact on (personalized) treatment strategies in PDAC and address how DNA barcoding technologies work and can be applied to study this clinically highly relevant question.

Altered Mitochondria Functionality Defines a Metastatic Cell State in Lung Cancer and Creates an Exploitable Vulnerability.

Lung cancer is a prevalent and lethal cancer type that leads to more deaths than the next four major cancer types combined. Metastatic cancer spread is responsible for most cancer-related deaths but the cellular changes that enable cancer cells to leave the primary tumor and establish inoperable and lethal metastases remain poorly understood. To uncover genes that are specifically required to sustain metastasis survival or growth, we performed a genome-scale pooled lentiviral-shRNA library screen in cells that represent nonmetastatic and metastatic states of lung adenocarcinoma. Mitochondrial ribosome and mitochondria-associated genes were identified as top gene sets associated with metastasis-specific lethality. Metastasis-derived cell lines in vitro and metastases analyzed ex vivo from an autochthonous lung cancer mouse model had lower mitochondrial membrane potential and reduced mitochondrial functionality than nonmetastatic primary tumors. Electron microscopy of metastases uncovered irregular mitochondria with bridging and loss of normal membrane structure. Consistent with these findings, compounds that inhibit mitochondrial translation or replication had a greater effect on the growth of metastasis-derived cells. Finally, mice with established tumors developed fewer metastases upon treatment with phenformin in vivo. These results suggest that the metastatic cell state in lung adenocarcinoma is associated with a specifically altered mitochondrial functionality that can be therapeutically exploited. SIGNIFICANCE: This study characterizes altered mitochondria functionality of the metastatic cell state in lung cancer and opens new avenues for metastasis-specific therapeutic targeting.

HER2 mediates clinical resistance to the KRASG12C inhibitor sotorasib, which is overcome by co-targeting SHP2.

INTRODUCTION: Mutant RAS guanosine triphosphate hydrolases (GTPases) are key oncogenic drivers in many cancers. The KRASG12C variant has recently become targetable by a new drug class specifically locking KRASG12C in its inactive guanosine diphosphate (GDP)-bound state. Clinical activity was demonstrated in patients with advanced lung cancers harbouring KRASG12C mutations but was limited by the development of resistance. METHODS: A biopsy from progressing lung cancer of a patient treated with the KRASG12C inhibitor sotorasib was obtained, and the underlying resistance factors were analysed. Mechanistic studies were performed in vitro and in vivo to uncover strategies to overcome resistance to KRASG12C inhibition. RESULTS: We demonstrated acquisition of HER2 copy number gain and KRASG12C mutation retention in the post-progression biopsy. To explore HER2 gain as the relevant resistance mechanism, we generated KRASG12C lung cancer models overexpressing HER2. MAPK pathway signalling remained active despite KRASG12C inhibitor treatment. Combined pharmacological inhibition of KRASG12C and SHP2 synergistically overcame HER2-mediated resistance in vitro and in vivo. CONCLUSIONS: These findings establish HER2 copy number gain as a clinically relevant mechanism of resistance to pharmacological KRASG12C inhibition that can be overcome by co-targeting SHP2.

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.

EF Bart's Disease with Coinheritance of Gγ-XmnI and Aγ-Globin Polymorphisms: A Case of Nontransfusion-Dependant Thalassemia.

EF Bart's disease is a rare form of nontransfusion-dependant thalassemia (NTDT) due to the coinheritance of homozygous hemoglobin E (ß E/ß E) genotype with hemoglobin H disease. These individuals are routinely found to have thalassemia intermedia with moderate anemia, increased hemoglobin Bart's and hemoglobin F on electrophoresis. The contribution of hemoglobin F-inducing polymorphisms in this disease has not been described previously. Here, we describe the hematological profile in a young child with coinheritance of Gγ-XmnI and Aγ-globin gene polymorphisms in EF Bart's disease. Interestingly, in this rare form of NTDT, normal HbF and elevated HbA2 were noted.

Functional screening identifies aryl hydrocarbon receptor as suppressor of lung cancer metastasis.

Lung cancer mortality largely results from metastasis. Despite curative surgery many patients with early-stage non-small cell lung cancer ultimately succumb to metastatic relapse. Current risk reduction strategies based on cytotoxic chemotherapy and radiation have only modest activity. Against this background, we functionally screened for novel metastasis modulators using a barcoded shRNA library and an orthotopic lung cancer model. We identified aryl hydrocarbon receptor (AHR), a sensor of xenobiotic chemicals and transcription factor, as suppressor of lung cancer metastasis. Knockdown of endogenous AHR induces epithelial-mesenchymal transition signatures, increases invasiveness of lung cancer cells in vitro and metastasis formation in vivo. Low intratumoral AHR expression associates with inferior outcome of patients with resected lung adenocarcinomas. Mechanistically, AHR triggers ATF4 signaling and represses matrix metalloproteinase activity, both counteracting metastatic programs. These findings link the xenobiotic defense system with control of lung cancer progression. AHR-regulated pathways are promising targets for innovative anti-metastatic strategies.