The role of subclonal
mutations, defined by a variant allele frequency of <20%, has not been addressed in acute myeloid leukemia yet. We, therefore, analyzed their prognostic value in a cohort of ...1,537 patients with newly diagnosed disease, prospectively treated within three trials of the "German-Austrian Acute Myeloid Leukemia Study Group". Mutational analysis was performed by targeted deep sequencing and patients with
mutations were categorized by their variant allele frequency into groups with frequencies >40%, 20%-40% and <20%. A total of 108
mutations were found in 98 patients (6.4%). Among these, 61 patients had variant allele frequencies >40%, 19 had variant allele frequencies between 20%-40% and 18 had frequencies <20%. Compared to specimens with clonal
mutations, those with subclonal ones showed significantly fewer complex karyotypes and chromosomal losses. In either
-mutated group, patients experienced significantly fewer complete responses (
<0.001) and had worse overall and event-free survival rates (
<0.0001). In Cox regression analyses adjusting for age, white blood cell count, cytogenetic risk and type of acute myeloid leukemia, the adverse prognostic effect of
mutations remained significant for all
-mutated subgroups. These data suggest that subclonal
mutations are a novel prognostic parameter in acute myeloid leukemia and emphasize the usefulness of next-generation sequencing technologies for risk stratification in this disorder. The study was registered at ClinicalTrials.gov with number NCT00146120.
Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These ...technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34
hematopoietic stem and progenitor cells (HSPCs) and human CD3
T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.
Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances ...in understanding mutant CALR, the detailed mechanisms are not fully elucidated and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo.
Our CRISPR/Cas9-mediated KI strategy enabled us to efficiently generate heterozygous CALR mutant human HSPCs. Correct integration and expression were confirmed on DNA, RNA, and protein level. Type-1 and type-2 CALR mutations led to TPO-independent growth, a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media, and a significantly enhanced megakaryocyte formation upon liquid culture differentiation. When transplanted into immunodeficient NSG mice, CALR mutated HSPCs showed robust long-term engraftment and increased CD41+ megakaryocyte progenitor formation in the bone marrow compared to CALR wild-type cells.
Our system allows for the first time to investigate human MPN pathogenesis prospectively and sheds light on the transforming mechanisms of mutant CALR in primary HSPCs. CALR transformed human HSPCs show increased megakaryocyte formation and platelet differentiation, thereby recapitulating the phenotype seen in ET and PMF patients. Since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant HSPCs provide a potent new strategy to generate MPN xenotransplants with defined genotypes.
Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) ...CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41
progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.
Introduction:
Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite ...recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype.
Methods:
We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential.
Results:
Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody.
Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation.
When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors.
To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF.
Conclusion:
Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies.
Greinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.
Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). ...Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays.
Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition.
Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed “Acute Myeloid Leukemia” among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells.
Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation.
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Ediriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.
Introduction
Acute myeloid leukemia (AML) with “TP53 mutation, chromosomal aneuploidy or both” has recently been described as a distinct AML subgroup exhibiting an exceedingly adverse prognosis. ...Therefore, testing for TP53 mutations has become an essential part of the European LeukemiaNet 2017 recommendations for risk classification. However, the incidence and pathogenic role of subclonal TP53 mutations defined by a variant allele frequency (VAF) <20% has not been addressed in this disorder yet.
Methods
We evaluated the prognostic value of TP53 VAFs in a cohort of 1537 patients with newly diagnosed AML, prospectively treated within 3 trials of the “German-Austrian AML Study Group”. Mutation testing was performed by targeted deep sequencing and patients with TP53 mutations were categorized into 3 groups defined by VAFs <20%, 20%-40% and >40%, respectively.
Results
A total of 108 TP53 mutations were identified in 98 patients (6.4%). Amongst those, 61 patients showed a VAF >40%, 19 a VAF between 20% and 40% and 18 a VAF <20%. In either group, the majority of mutations were missense substitutions (VAF >40%, 49/61 80%; 20%-40%, 17/19 89%; <20%, 17/18 94%) and were located in the DNA binding domain of the gene (VAF >40%, 49/61 80%; 20%-40%, 16/19 84%; <20%, 16/18 89%). Compared to AML with clonal TP53 mutations, those with subclonal ones showed significantly fewer complex karyotypes and chromosomal losses. The number of cooperating driver mutations was low with 0-5 per case and did not differ between the 3 TP53 mutated groups. The complete remission (CR) rate was significantly inferior in the TP53 mutated cohort (48.0% versus 84.9% for TP53 wild-type patients, P <0.001) but equally distributed among the VAF-based subgroups (TP53 VAF >40%, 42.6%; 20%-40%, 57.9%; <20%, 55.6%; P=0.424). The estimated median overall survival (OS) for all 1537 AML patients was 28.1 months (95% CI, 24.3-33.5) but differed substantially between TP53 wild-type and TP53 mutated patients (33.6 months 95%CI, 28.4-45.0 versus 6.5 months 95%CI, 5.0-8.2). As depicted in Figure 1, OS rates were comparably low in all TP53 mutated subgroups (TP53 VAF >40%, 5.8 months; 20%-40%, 6.9 months; <20%, 6.9 months).The median estimated event-free survival (EFS) for all 1537 AML patients was 15.0 months (95% CI, 13.6-16.5) with that for TP53 wild-type patients being 16.5 months (95% CI, 15.0-18.2) and for TP53 mutated patients 5.7 months (95% CI, 4.3-7.4). Median EFS rates were comparably low in all TP53 mutated subgroups (TP53 VAF >40%, 5.2 months; 20%-40%, 6.9 months; <20%, 6.5 months). In Cox regression analyses adjusting for age, white blood cell count, cytogenetic risk and type of AML, the adverse prognostic effect of TP53 mutations remained significant, with subclonal mutations showing the strongest impact (hazard ratio, 3.71 95% CI, 2.11-6.51 for OS).
Conclusion
In our study on a large cohort of AML patients, TP53 mutated subclones defined by VAF <20% account for ~18% of all TP53 mutated AML cases. We here provide evidence that subclonal TP53 mutations are associated with a comparable negative impact on prognosis as clonal TP53 mutations. These findings may have implications for TP53 screening methods and for future risk stratification in AML.
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No relevant conflicts of interest to declare.
Abstract Although computer-aided engineering (CAE) software has been used for many years in the plastic industry, identifying the most appropriate mesh geometry and density remains a challenge. It ...can affect the accuracy of the simulation, the time and the costs. The evaluation of the most suitable mesh is not easy because the difficulties to obtain the real the values of the pressure and temperature inside the mold. The current work investigates this issue. A mold was manufactured and sensors were installed in its interior. CAE simulations using different mesh geometries and densities were evaluated against the experimental data. The results showed that the computational time was mostly influenced by the mesh geometry. The use of 2D mesh and lower density can lead to a faster and more precise simulation of pressure inside the mold and 3D mesh with lower density can provide a faster and precise simulation of the temperature.
The injection molding process is very sensitive to ordinary environmental alterations, as the numerical simulation is limited to within one injection cycle, and it cannot predict transient regimes. ...The present study presents a new approach based on SARIMAX models developed to predict the temperature and pressure inside the mold cavity. The proposed approach was developed in Python language, and it can identify the behavior of the process, allowing preventive actions. Experimental data of temperature and pressure obtained in real‐time inside an injection mold were accessed to use and to validate the proposed model. The results showed its efficiency and its high accuracy for predicting variations in temperature and pressure inside the mold, even when using a small number of samples to be trained. The proposed model can be very useful for monitoring the production of mechanical parts, under an Industry 4.0 environment. For future works, the model enables a contribution toward digital twins of a molded part, considering all the alteration on the parts' properties due to the disturbance on the injection molding process. Furthermore, it lays the groundwork for a new injection machine control system architecture.
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