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Development ◽  
2021 ◽  
Vol 148 (24) ◽  
Author(s):  
Carrie M. Spratford ◽  
Lauren M. Goins ◽  
Fangtao Chi ◽  
Juliet R. Girard ◽  
Savannah N. Macias ◽  
...  

ABSTRACT Genetic and genomic analysis in Drosophila suggests that hematopoietic progenitors likely transition into terminal fates via intermediate progenitors (IPs) with some characteristics of either, but perhaps maintaining IP-specific markers. In the past, IPs have not been directly visualized and investigated owing to lack of appropriate genetic tools. Here, we report a Split GAL4 construct, CHIZ-GAL4, that identifies IPs as cells physically juxtaposed between true progenitors and differentiating hemocytes. IPs are a distinct cell type with a unique cell-cycle profile and they remain multipotent for all blood cell fates. In addition, through their dynamic control of the Notch ligand Serrate, IPs specify the fate of direct neighbors. The Ras pathway controls the number of IP cells and promotes their transition into differentiating cells. This study suggests that it would be useful to characterize such intermediate populations of cells in mammalian hematopoietic systems.


2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amna Yaqub ◽  
Harikrishna Harikrishna Nakshatri2

Background and Hypothesis: The RAS and PI3K-AKT-mTOR signaling pathways are often dysregulated in cancer. RAS pathway alterations, however, are more common in breast cancer metastasis. The laboratory’s recently developed model system demonstrated the ability of RAS but not PIK3CA-induced signals in promoting metastasis of breast cancer. Unbiased kinome analyses of isogenic RAS-transformed primary tumor and metastatic cells and PIK3CA-transformed primary tumor cells enabled identification of RAS-activated kinome, which included FER, PAK4, LIMK1, PIK3CD and Casein Kinase 2 (CK2). We hypothesized that therapeutic targeting of these kinases may reduce breast cancer metastasis. As a proof-of-principle, the effect of the CK2 inhibitor Silmitasertib, which is in clinical trial for COVID-19 and refractory multiple myeloma, was tested. Experimental Design: The study included four isogenic cell lines: “normal” (KTB34-hTERT), PIK3CA-transformed (TKTB34-PIK3CA), RAS-transformed (TKTB34-RAS), and RAS-transformed cells metastasized to lungs (MKTB34-RAS). Active kinomes in these cells were identified using phospho-proteomics and functional kinome profiling using multiplexed kinase inhibitor beads. Expression levels of FER, PAK4, LIMK1, and PIK3CD kinases were compared through Western Blot using the phospho-antibodies as an indicator of kinase activation. Sensitivity to Silmitasertib was measured using the BrdU Cell Proliferation Assay. Results: FER, PAK4, LIMK1, and PIK3CD were all overexpressed in the TKTB34-RAS and MKTB34-RAS cells compared to KTB34-hTERT and TKTB34-PIK3CA cells. The tested concentration range for Silmitasertib (500 nM to 5 µM) was ineffective in killing the RAS-transformed cells and was overly toxic to “normal” cells. Conclusion and Potential Impact: FER, PAK4, LIMK1, PIK3CD, and CK2 are potential therapeutic targets for breast cancer metastasis. However, Silmitasertib may not be a good candidate as it is more toxic to “normal” cells compared to cancer cells. The isogenic “normal” and transformed cell line model system described here may help to discover new targets and drugs that kill cancer but not normal cells.


2021 ◽  
Author(s):  
Yandan Yang ◽  
Thomas Oellerich ◽  
Ping Chen ◽  
Arnold Bolomsky ◽  
Michele Ceribelli ◽  
...  

Oncogenic mutations within the RAS pathway are common in multiple myeloma (MM), an incurable malignancy of plasma cells. However, the mechanisms of pathogenic RAS signaling in this disease remain enigmatic and difficult to inhibit therapeutically. We employed an unbiased proteogenomic approach to dissect RAS signaling in MM by combining genome-wide CRISPR-Cas9 screening with quantitative mass spectrometry focused on RAS biology. We discovered that mutant isoforms of RAS organized a signaling complex with the amino acid transporter, SLC3A2, and MTOR on endolysosomes, which directly activated mTORC1 by co-opting amino acid sensing pathways. MM tumors with high expression of mTORC1-dependent genes were more aggressive and enriched in RAS mutations, and we detected interactions between RAS and MTOR in MM patient tumors harboring mutant RAS isoforms. Inhibition of RAS-dependent mTORC1 activity synergized with MEK and ERK inhibitors to quench pathogenic RAS signaling in MM cells. This study redefines the RAS pathway in MM and provides a mechanistic and rational basis to target this novel mode of RAS signaling.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Olusegun Isaac Alatise ◽  
Gregory C. Knapp ◽  
Avinash Sharma ◽  
Walid K. Chatila ◽  
Olukayode A. Arowolo ◽  
...  

AbstractUnderstanding the molecular and phenotypic profile of colorectal cancer (CRC) in West Africa is vital to addressing the regions rising burden of disease. Tissue from unselected Nigerian patients was analyzed with a multigene, next-generation sequencing assay. The rate of microsatellite instability is significantly higher among Nigerian CRC patients (28.1%) than patients from The Cancer Genome Atlas (TCGA, 14.2%) and Memorial Sloan Kettering Cancer Center (MSKCC, 8.5%, P < 0.001). In microsatellite-stable cases, tumors from Nigerian patients are less likely to have APC mutations (39.1% vs. 76.0% MSKCC P < 0.001) and WNT pathway alterations (47.8% vs. 81.9% MSKCC, P < 0.001); whereas RAS pathway alteration is more prevalent (76.1% vs. 59.6%, P = 0.03). Nigerian CRC patients are also younger and more likely to present with rectal disease (50.8% vs. 33.7% MSKCC, P < 0.001). The findings suggest a unique biology of CRC in Nigeria, which emphasizes the need for regional data to guide diagnostic and treatment approaches for patients in West Africa.


Haematologica ◽  
2021 ◽  
Author(s):  
Thomas Creasey ◽  
Emilio Barretta ◽  
Sarra L. Ryan ◽  
Ellie Butler ◽  
Amy A Kirkwood ◽  
...  

Despite being predominantly a childhood disease, the incidence of ALL has a second peak in adults aged 60 years and over. These older adults fare extremely poorly with existing treatment strategies and very few studies have undertaken a comprehensive genetic and genomic characterisation to improve prognosis in this age group. We performed cytogenetic, single nucleotide polymorphism (SNP) array and next generation sequencing (NGS) analyses on samples from 210 patients aged ≥60 years from the UKALL14 and UKALL60+ clinical trials. BCR-ABL1 positive disease was present in 26% (55/210) of patients, followed by low hypodiploidy/near triploidy in 13% (28/210). Cytogenetically cryptic rearrangements in CRLF2, ZNF384 and MEF2D were detected in 5%, 1% and 1% of patients respectively. Copy number abnormalities were common and deletions in ALL driver genes were seen in 77% of cases. IKZF1 deletion was present in 51% (40/78) of samples tested and the IKZF1plus profile identified in over a third (28/77) of BCP-ALL cases. The genetic good risk abnormalities high hyperdiploidy (n=2), ETV6-RUNX1 (no cases) and ERG deletion (no cases) were exceptionally rare in this cohort. RAS pathway mutations were seen in 17% (4/23) of screened samples. KDM6A abnormalities, including biallelic deletions, were discovered in 5% (4/78) of SNP array and 9% (2/23) of NGS samples, and represent a novel, potentially therapeutically actionable lesions using EZH2 inhibitors. Outcome remained poor with five-year event-free (EFS) and overall survival (OS) rates of 17% and 24% respectively across the cohort indicating a need for novel therapeutic strategies.


Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1177
Author(s):  
Bhagyashri Joshi ◽  
Ganesh Wagh ◽  
Harmandeep Kaur ◽  
Chinmoy Patra

Hypertension, a common chronic condition, may damage multiple organs, including the kidney, heart, and brain. Thus, it is essential to understand the pathology upon ectopic activation of the molecular pathways involved in mammalian hypertension to develop strategies to manage hypertension. Animal models play a crucial role in unraveling the disease pathophysiology by allowing incisive experimental procedures impossible in humans. Zebrafish, a small freshwater fish, have emerged as an important model system to study human diseases. The primary effector, Angiotensin II of the RAS pathway, regulates hemodynamic pressure overload mediated cardiovascular pathogenesis in mammals. There are various established mammalian models available to study pathophysiology in Angiotensin II-induced hypertension. Here, we have developed a zebrafish model to study pathogenesis by Angiotensin II. We find that intradermal Angiotensin II injection every 12 h can induce cardiac remodeling in seven days. We show that Angiotensin II injection in adult zebrafish causes cardiomyocyte hypertrophy and enhances cardiac cell proliferation. In addition, Angiotensin II induces ECM protein-coding gene expression and fibrosis in the cardiac ventricles. Thus, this study can conclude that Angiotensin II injection in zebrafish has similar implications as mammals, and zebrafish can be a model to study pathophysiology associated with AngII-RAS signaling.


2021 ◽  
Vol 22 (22) ◽  
pp. 12142
Author(s):  
Soonsil Hyun ◽  
Dongyun Shin

Drug resistance continues to be a major problem associated with cancer treatment. One of the primary causes of anticancer drug resistance is the frequently mutated RAS gene. In particular, considerable efforts have been made to treat KRAS-induced cancers by directly and indirectly controlling the activity of KRAS. However, the RAS protein is still one of the most prominent targets for drugs in cancer treatment. Recently, novel targeted protein degradation (TPD) strategies, such as proteolysis-targeting chimeras, have been developed to render “undruggable” targets druggable and overcome drug resistance and mutation problems. In this study, we discuss small-molecule inhibitors, TPD-based small-molecule chemicals for targeting RAS pathway proteins, and their potential applications for treating KRAS-mutant cancers. Novel TPD strategies are expected to serve as promising therapeutic methods for treating tumor patients with KRAS mutations.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 207-207
Author(s):  
Daelynn R Buelow ◽  
Bhavana Bhatnagar ◽  
Jae Yoon Jeon ◽  
Megan Zavorka Thomas ◽  
Eric Eisenmann ◽  
...  

Abstract While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. Disclosures Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4643-4643
Author(s):  
Lisa P. Chu ◽  
Malathi Kandarpa ◽  
Dan Robinson ◽  
Yi-Mi Wu ◽  
Moshe Talpaz ◽  
...  

Abstract Purpose: Myelofibrosis (MF) is a clonal stem-cell derived BCR-ABL negative myeloproliferative neoplasm characterized by atypical megakaryocytic proliferation leading to bone marrow reticulin and collagen fibrosis. About 90% of patients with MF harbor somatic driver mutations that constitutively activate the JAK-STAT pathway. There have been other high molecular risk genes identified (ASXL1, EZH2, IDH1, IDH2, SRSF2) that predict inferior survival and have been added to prognostic scoring systems (Cazzola et al, Blood 2014). RAS pathway activating mutations are common in myeloid malignancies. Mutations in NRAS or KRAS themselves have been reported in ~5% of MF cases, and are associated with more proliferative disease behavior, decreased overall survival, and decreased chances of response to JAK inhibitor therapy (Coltro et al, Blood Adv 2020; Santos et al, Leukemia 2020). Downstream mutations in other RAS pathway genes such as NF1, BRAF, CBL, or PTPN11 are relatively common in MF, although their impact on disease behavior or response to therapy is not yet well characterized. Here, we aim to characterize clinical features of RAS pathway activated MF, describe response to therapies including JAK inhibitors, and determine impact on patient outcomes. Methods: We retrospectively analyzed the medical records of 118 consecutive adult patients with myelofibrosis who were seen at our tertiary referral center between July 2011 and July 2021 and who had next generation sequencing (NGS) performed - either MiOncoseq (Roychowdhury et al, Sci Transl Med 2011) or Tempus myeloid NGS. We grouped patients by those with mutated NRAS and/or KRAS (n=27) and those without mutated NRAS or KRAS but with another RAS pathway activating mutation (CBL, NF1, BRAF, PTPN11; n=12). Results: Of our cohort with MF, 39 (33%) harbored a RAS pathway activating mutation: NRAS, n=17; KRAS, n=15; CBL, n=12; NF1, n=6; BRAF, n=2; PTPN11, n=4. Fifteen (38%) patients had at least 2 different RAS pathway mutations. All of these patients had a JAK2 (n=29), CALR (n=6) or MPL (n=5) mutation in addition to their RAS pathway mutation. Ruxolitinib was the most common first-line MF directed therapy in 27 patients (69%); other less common first-line treatments included clinical trials containing a JAK inhibitor (5), hydroxyurea (3), interferon alpha (2), or danazol (2). With first-line therapy, overall response rate by IWG-MRT criteria was 49% (n=19), 16 (41%) had stable disease, and 4 (10%) had resistant disease. There was no statistical difference in type of response to first-line ruxolitinib between the NRAS/KRAS mutated group and the other RAS pathway mutated group (p=0.8). Twenty-seven patients went on to receive second-line therapy - 14 patients on a clinical trial with a JAK inhibitor. Twelve patients (31%) received at least 3 unique lines of treatment. After median follow-up of 69 months, 13 (33%) were alive and 26 (67%) had died, with 22 of these deaths related to their myelofibrosis diagnosis. Median overall survival was 74.7 months, 65.3 months in the NRAS/KRAS mutated group, and 76.2 months in the other RAS pathway mutated group, with no difference in overall survival between the two groups (p=0.72). Twenty patients (74%) from the NRAS/KRAS mutated group had a cause of death related to MF compared to 6 patients (50%) in the other group. Overall, 12 patients (31%) had leukemic transformation, 9 (33%) in the NRAS/KRAS mutated group and 3 (25%) in the other RAS pathway mutated group (p=0.72). Conclusion: Most patients with RAS pathway mutations presented with intermediate risk clinical characteristics (based on the DIPSS risk status), splenomegaly, and constitutional symptoms. Among those treated first-line with JAK inhibitors, about 50% of patients responded to therapy, similar to prior studies looking at JAK inhibitors in MF, though duration of response was short in both of the RAS activated subgroups (24 months and 28 months). In comparing patients with direct NRAS/KRAS mutations to those with other RAS pathway mutations, there was no difference between risk status at diagnosis, treatment responses overall and to ruxolitinib specifically, or overall survival. While our sample size was limited, our data suggests that patients with direct NRAS/KRAS mutations had similar clinical features and outcomes compared to those with other RAS pathway mutations. Further studies with larger cohorts are warranted to help predict response to JAK inhibitors. Figure 1 Figure 1. Disclosures Talpaz: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Imago: Consultancy; Constellation: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Grant/research support .


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