bims-tremyl 2022-10-16 papers

on Therapy resistance biology in myeloid leukemia

Issue of 2022–10–16
thirty-one papers selected by
Paolo Gallipoli, Barts Cancer Institute, Queen Mary University of London

Autophagy inhibition impairs leukemia stem cell function in FLT3-ITD AML but has antagonistic interactions with tyrosine kinase inhibition.
TP53-Mutated Myelodysplastic Syndrome and Acute Myeloid Leukemia: Biology, Current Therapy, and Future Directions.
Concomitant targeting of FLT3 and BTK overcomes FLT3 inhibitor resistance in acute myeloid leukemia through inhibition of autophagy.
Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia.
Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance.
Early mortality risk with non-intensive acute myeloid leukemia (AML) therapies: analysis of 1336 patients from MRC/NCRI and SWOG.
Acquired mutations in BAX confer resistance to BH3-mimetic therapy in Acute Myeloid Leukemia.
Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.
Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia.
The Cup Runneth Over: Treatment Strategies for Newly Diagnosed Acute Myeloid Leukemia.
Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.
Incidence of Invasive Fungal Infections in Patients With Previously Untreated Acute Myeloid Leukemia Receiving Venetoclax and Azacitidine.
Multiomics data integration to reveal chromatin remodeling and reorganization induced by gene mutational synergy.
Impact of Clonal Hematopoiesis in Patients With Cardiogenic Shock Complicating Acute Myocardial Infarction.
Dasatinib plus Peg-Interferon alpha 2b combination in newly diagnosed chronic phase chronic myeloid leukaemia: Results of a multicenter phase 2 study (DASA-PegIFN study).
LYL1 facilitates AETFC assembly and gene activation by recruiting CARM1 in t(8;21) AML.
Clonal hematopoiesis in patients with stem cell mobilization failure: a nested case-control study.
Analysis of Disparities in Time to Allogeneic Transplantation in Adults with Acute Myelogenous Leukemia.
Comparison of the revised 4th (2016) and 5th (2022) editions of the World Health Organization classification of myelodysplastic neoplasms.
Current status of phase 3 clinical trials in high-risk myelodysplastic syndromes: pitfalls and recommendations.
Pathogenicity and impact of HLA class I alleles in aplastic anemia patients of different ethnicities.
DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.
Safety, outcomes and T cell characteristics in patients with relapsed or refractory MDS or CMML treated with atezolizumab in combination with guadecitabine.
Isolation, culture, and immunophenotypic analysis of bone marrow HSPCs from patients with myelodysplastic syndromes.
Incidence of adverse effects in patients receiving ponatinib with concomitant azole antifungals.
c-Mpl-del, a c-Mpl alternative splicing isoform, promotes AMKL progression and chemoresistance.
RUNX1-deficient human megakaryocytes demonstrate thrombopoietic, and platelet half-life and functional defects.
The new diagnostic criteria for myelodysplasia-related acute myeloid leukemia is useful for predicting clinical outcome: comparison of the 4th and 5th World Health Organization classifications.
Donor lymphocyte infusions after haploidentical stem cell transplantation with PTCY: A study on behalf of the EBMT cellular therapy & immunobiology working party.
HSC derived fatty acid oxidation in steady state and stressed haematopoiesis.
Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

Leukemia. 2022 Oct 11.

Autophagy inhibition impairs leukemia stem cell function in FLT3-ITD AML but has antagonistic interactions with tyrosine kinase inhibition.

Shaowei Qiu, Harish Kumar, Chengcheng Yan, Hui Li, Andrew J Paterson, Nicholas R Anderson, Jianbo He, Jing Yang, Min Xie, David K Crossman, Rui Lu, Robert S Welner, Ravi Bhatia.

The FLT3-ITD mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) demonstrate clinical efficacy but fail to target leukemia stem cells (LSC) and do not generate sustained responses. Autophagy is an important cellular stress response contributing to hematopoietic stem cells (HSC) maintenance and promoting leukemia development. Here we investigated the role of autophagy in regulating FLT3-ITD AML stem cell function and response to TKI treatment. We show that autophagy inhibition reduced quiescence and depleted repopulating potential of FLT3-ITD AML LSC, associated with mitochondrial accumulation and increased oxidative phosphorylation. However, TKI treatment reduced mitochondrial respiration and unexpectedly antagonized the effects of autophagy inhibition on LSC attrition. We further show that TKI-mediated targeting of AML LSC and committed progenitors was p53-dependent, and that autophagy inhibition enhanced p53 activity and increased TKI-mediated targeting of AML progenitors, but decreased p53 activity in LSC and reduced TKI-mediated LSC inhibition. These results provide new insights into the role of autophagy in differentially regulating AML stem and progenitor cells, reveal unexpected antagonistic effects of combined oncogenic tyrosine kinase inhibition and autophagy inhibition in AML LSC, and suggest an alternative approach to target AML LSC quiescence and regenerative potential.

DOI: https://doi.org/10.1038/s41375-022-01719-6
Cancer Discov. 2022 Oct 11. OF1-OF14

TP53-Mutated Myelodysplastic Syndrome and Acute Myeloid Leukemia: Biology, Current Therapy, and Future Directions.

Naval G Daver, Abhishek Maiti, Tapan M Kadia, Paresh Vyas, Ravindra Majeti, Andrew H Wei, Guillermo Garcia-Manero, Charles Craddock, David A Sallman, Hagop M Kantarjian.

TP53-mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) form a distinct group of myeloid disorders with dismal outcomes. TP53-mutated MDS and AML have lower response rates to either induction chemotherapy, hypomethylating agent-based regimens, or venetoclax-based therapies compared with non-TP53-mutated counterparts and a poor median overall survival of 5 to 10 months. Recent advances have identified novel pathogenic mechanisms in TP53-mutated myeloid malignancies, which have the potential to improve treatment strategies in this distinct clinical subgroup. In this review, we discuss recent insights into the biology of TP53-mutated MDS/AML, current treatments, and emerging therapies, including immunotherapeutic and nonimmune-based approaches for this entity.
SIGNIFICANCE: Emerging data on the impact of cytogenetic aberrations, TP53 allelic burden, immunobiology, and tumor microenvironment of TP53-mutated MDS and AML are further unraveling the complexity of this disease. An improved understanding of the functional consequences of TP53 mutations and immune dysregulation in TP53-mutated AML/MDS coupled with dismal outcomes has resulted in a shift from the use of cytotoxic and hypomethylating agent-based therapies to novel immune and nonimmune strategies for the treatment of this entity. It is hoped that these novel, rationally designed combinations will improve outcomes in this area of significant unmet need.

DOI: https://doi.org/10.1158/2159-8290.CD-22-0332
Haematologica. 2022 Oct 13.

Concomitant targeting of FLT3 and BTK overcomes FLT3 inhibitor resistance in acute myeloid leukemia through inhibition of autophagy.

Weiguo Zhang, Guopan Yu, Hongying Zhang, Mahesh Basyal, Charlie Ly, Bin Yuan, Vivian Ruvolo, Sujan Piya, Seemana Bhattacharya, Qi Zhang, Gautam Borthakur, Venkata Battula, Marina Konopleva, William G Rice, Michael Andreeff.

Strategies to overcome resistance to FMS-like tyrosine kinase 3 (FLT3)-targeted therapy in acute myeloid leukemia (AML) are urgently needed. We identify autophagy as one of the resistance mechanisms, induced by hypoxia and the bone marrow (BM) microenvironment via Bruton's tyrosine kinase (BTK) activation. Suppressing autophagy/BTK sensitized FLT3-mutated AML to FLT3 inhibitor-induced apoptosis. Further, co-targeting FLT3/BTK/Aurora kinases (AURKs) with a novel multi-kinase inhibitor CG-806 (luxeptinib) induced profound apoptosis induction in FLT3-mutated AML by co-suppressing FLT3/BTK, antagonizing autophagy, and causing leukemia cell death in FLT3 wild-type AML by AURK-mediated G2/M arrest and polyploidy, in addition to FLT3 inhibition. Thus, CG-806 exerted profound anti-leukemia activity against AMLs regardless of FLT3 mutation status. CG-806 further significantly reduced AML burden and extended survival in an in vivo PDX leukemia murine model of FLT3 inhibitorresistant FLT3-ITD/TKD double mutant primary AML. Taken together, CG-806 exerts a unique mechanistic action and pre-clinical activity, suggesting further development in FLT3 wild-type and mutant AML.

DOI: https://doi.org/10.3324/haematol.2022.280884
Blood Adv. 2022 Oct 14. pii: bloodadvances.2022008141. [Epub ahead of print]

Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia.

Catia Simoes, Maria Carmen Chillon, David Martínez-Cuadrón, Maria José Calasanz, María-Belén Vidriales, Iria Vazquez, Montserrat Hernández-Ruano, Beñat Ariceta, Paula Aguirre-Ruiz, Leire Burgos, Diego Alignani, Sarai Sarvide, Sara Villar, Ana Alfonso, Felipe Prosper, Rosa Ayala, Joaquin Martínez-López, Juan Miguel Bergua Burgues, Susana Vives, Jose Antonio Perez-Simon, María García-Fortes, Teresa Bernal Del Castillo, Mercedes Colorado, Mayte Olave, Juan I Rodríguez-Gutiérrez, Jorge Labrador, Marcos González, Jesus San-Miguel, Miguel A Sanz, Pau Montesinos, Bruno Paiva.

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workup. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly-diagnosed AML patients. Presence of dysplasia according to MFC and WHO criteria had no prognostic value in the elderly. NGS of dysplastic cells and blasts isolated at diagnosis identified three evolutionary patterns: stable (n=12/21), branching (n=4/21) and clonal evolution (n=5/21). In patients achieving complete response, integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in approximately 80% of newly diagnosed AML patients, using techniques other than single-cell multiomics.

DOI: https://doi.org/10.1182/bloodadvances.2022008141
Cancer Discov. 2022 Oct 12. pii: CD-21-1661. [Epub ahead of print]

Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance.

Junhua Lyu, Yuxuan Liu, Lihu Gong, Mingyi Chen, Yazan F Madanat, Yuannyu Zhang, Feng Cai, Zhimin Gu, Hui Cao, Pranita Kaphle, Yoon Jung Kim, Fatma N Kalkan, Helen Stephens, Kathryn E Dickerson, Min Ni, Weina Chen, Prapti Patel, Alice S Mims, Uma Borate, Amy Burd, Sheng F Cai, C Cameron Yin, M James You, Stephen S Chung, Robert H Collins, Ralph J DeBerardinis, Xin Liu, Jian Xu.

Mutations in IDH genes occur frequently in acute myeloid leukemia (AML) and other human cancers to generate the oncometabolite R-2HG. Allosteric inhibition of mutant IDH suppresses R-2HG production in a subset of AML patients; however, acquired resistance emerges as a new challenge and the underlying mechanisms remain incompletely understood. Here we establish isogenic leukemia cells containing common IDH oncogenic mutations by CRISPR base editing. By mutational scanning of IDH single-amino acid variants in base-edited cells, we describe a repertoire of IDH second-site mutations responsible for therapy resistance through disabling uncompetitive enzyme inhibition. Recurrent mutations at NADPH binding sites within IDH heterodimers act in cis or trans to prevent the formation of stable enzyme-inhibitor complexes, restore R-2HG production in the presence of inhibitors, and drive therapy resistance in IDH-mutant AML cells and patients. We therefore uncover a new class of pathogenic mutations and mechanisms for acquired resistance to targeted cancer therapies.

DOI: https://doi.org/10.1158/2159-8290.CD-21-1661
Leuk Lymphoma. 2022 Oct 13. 1-3

Early mortality risk with non-intensive acute myeloid leukemia (AML) therapies: analysis of 1336 patients from MRC/NCRI and SWOG.

Megan Othus, Ian Thomas, Xu Wang, Cono Ariti, Priyanka Mehta, Mia Sydenham, Robert K Hills, Alan K Burnett, Sucha Nand, Sarit Assouline, Laura C Michaelis, Harry P Erba, Nigel Russell, Kathleen F Kerr, Roland B Walter, Mike Dennis.

DOI: https://doi.org/10.1080/10428194.2022.2131416
Blood. 2022 Oct 11. pii: blood.2022016090. [Epub ahead of print]

Acquired mutations in BAX confer resistance to BH3-mimetic therapy in Acute Myeloid Leukemia.

Donia M Moujalled, Fiona C Brown, Chong Chyn Chua, Michael A Dengler, Giovanna Pomilio, Natasha S Anstee, Veronique Litalien, Ella R Thompson, Thomas David Morley, Sarah MacRaild, Ing Soo Tiong, Rhiannon Morris, Karen Dun, Adrian Carl Zordan, Jaynish S Shah, Sébastien Banquet, Ensar Halilovic, Erick J Morris, Marco J Herold, Guillaume L Lessene, Jerry M Adams, David Ching Siang Huang, Andrew W Roberts, Piers Blombery, Andrew H Wei.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or pre-leukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represents a novel mechanism of resistance to BH3-mimetics and a potential barrier to longer-term efficacy of drugs targeting BCL-2 in AML.

DOI: https://doi.org/10.1182/blood.2022016090
Clin Cancer Res. 2022 Oct 14. pii: CCR-22-0978. [Epub ahead of print]

Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies.

Yifan Zhai, Qiuqiong Tang, Douglas D Fang, Jing Deng, Kaixiang Zhang, Qixin Wang, Yan Yin, Chengcheng Fu, Sheng-Li Xue, Na Li, Feng Zhou, Dajun Yang.

PURPOSE: Despite approval of BCL-2 inhibitor venetoclax for certain hematologic malignancies, its broader clinical benefit is curtailed by resistance. Our study aimed to determine if treatment with novel anticancer agents targeting BCL-2 and MDM2 could overcome venetoclax resistance in preclinical models.
EXPERIMENTAL DESIGN: Venetoclax-sensitive and venetoclax-resistant acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) cells and xenograft models were used to evaluate antitumor effects and underlying mechanisms associated with combined BCL-2 inhibitor lisaftoclax (APG-2575) and MDM2 inhibitor alrizomadlin (APG-115).
RESULTS: The combination exhibited synergistic antiproliferative and apoptogenic activities in TP53 wild-type (TP53WT) AML cell lines in vitro. This synergy was further exemplified by deep antitumor responses and prolonged survival in AML cell line-derived - and patient-derived xenograft (CDX and PDX) models. Interestingly, the combination treatment resensitized (to apoptosis) venetoclax-resistant cellular and mouse models established via chronic drug exposure or genetically engineered with clinically relevant BCL-2 gene mutations. Synergistic effects in reducing cellular viability and proliferation were also demonstrated in primary samples of patients with venetoclax-resistant AML treated with lisaftoclax and alrizomadlin ex vivo. Mechanistically, alrizomadlin likely primes cancer cells to BCL-2 inhibition-induced cellular apoptosis by downregulating expression of antiapoptotic proteins MCL-1 and BCL-xL and upregulating pro-death protein BAX.
CONCLUSIONS: Lisaftoclax in combination with alrizomadlin overcomes venetoclax resistance mediated by various mechanisms, including BCL-2 mutations. In addition, we posit further, putative molecular mechanisms. Our data rationalize clinical development of this treatment combination in patients with diseases that are insensitive or resistant to venetoclax.

DOI: https://doi.org/10.1158/1078-0432.CCR-22-0978
Biochem Pharmacol. 2022 Oct 05. pii: S0006-2952(22)00377-X. [Epub ahead of print]205 115283

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia.

Katie Hege Hurrish, Xinan Qiao, Xinyu Li, Yongwei Su, Jenna Carter, Jun Ma, Hasini A Kalpage, Maik Hüttemann, Holly Edwards, Guan Wang, Seongho Kim, Alan Dombkowski, Xun Bao, Jing Li, Jeffrey W Taub, Yubin Ge.

  Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

Keywords:  Acute myeloid leukemia; CUDC-907; Cytarabine (AraC) resistance; Oxidative phosphorylation; Venetoclax; c-Myc

DOI:  https://doi.org/10.1016/j.bcp.2022.115283
JCO Oncol Pract. 2022 Oct 12. OP2200342

The Cup Runneth Over: Treatment Strategies for Newly Diagnosed Acute Myeloid Leukemia.

Jennifer H Cooperrider, Navika Shukla, Mariam T Nawas, Anand Ashwin Patel.

Since 2017, the number of agents for acute myeloid leukemia (AML) has rapidly expanded. Given the increased therapeutic options, better identification of high-risk subsets of AML and more refined approaches to patient fitness assessment, the decisions surrounding selection of intensive chemotherapy versus lower-intensity treatment have grown increasingly more nuanced. In this review, we present available data for both standard and investigational approaches in the initial treatment of AML using an intensive chemotherapy backbone or a lower-intensity approach. We summarize management strategies in newly diagnosed secondary AML, considerations around allogeneic stem-cell transplantation, and the role of maintenance therapy. Finally, we highlight important areas of future investigation and novel agents that may hold promise in combination with standard therapies.

DOI: https://doi.org/10.1200/OP.22.00342
Nat Chem Biol. 2022 Oct 13.

Targeting lipid-protein interaction to treat Syk-mediated acute myeloid leukemia.

Indira Singaram, Ashutosh Sharma, Shashank Pant, Muyun Lihan, Mi-Jeong Park, Melissa Pergande, Pawanthi Buwaneka, Yusi Hu, Nadim Mahmud, You-Me Kim, Stephanie Cologna, Vladimir Gevorgyan, Irum Khan, Emad Tajkhorshid, Wonhwa Cho.

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing new small molecule drugs.

DOI: https://doi.org/10.1038/s41589-022-01150-z
Open Forum Infect Dis. 2022 Oct;9(10): ofac486

Incidence of Invasive Fungal Infections in Patients With Previously Untreated Acute Myeloid Leukemia Receiving Venetoclax and Azacitidine.

Alexander Zhang, Tanner Johnson, Diana Abbott, Tanit Phupitakphol, Jonathan A Gutman, Daniel A Pollyea, Yiannis Koullias.

   Background: Acute myeloid leukemia (AML) is associated with poor prognosis, particularly in elderly patients with comorbidities. Combining azacitidine (AZA) with BCL-2 inhibitor venetoclax (VEN) demonstrated significant improvement in outcomes for newly-diagnosed AML patients compared to AZA alone. However, this regimen is myelosuppressive, and the incidence of invasive fungal infections (IFIs) and impact of antifungal prophylaxis are not well defined.
Methods: This retrospective cohort study evaluated newly-diagnosed AML patients treated with VEN/AZA at the University of Colorado Hospital from January 2014 to August 2020. Patients with history of prior IFI were excluded. Primary outcome was IFI incidence during VEN/AZA therapy. χ2 and Fisher exact tests assessed the impact of patient demographics, AML-specific risk factors, and receipt of antifungal prophylaxis on IFI incidence.
Results: 144 VEN/AZA-treated AML patients were included in the study. 25 (17%) patients developed IFI: 8% (n = 2) "proven," 24% (n = 6) "probable," and 68% (n = 17) "possible" per European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium criteria. There was no statistically significant association between IFI incidence with age, sex, or European LeukemiaNet classification. 10 patients received antifungal prophylaxis; none developed IFI. IFI incidence rate per 1000 patient-days was greatest 0-9 days after starting VEN/AZA, at 8.39.
Conclusions: Incidence of "proven" and "probable" IFI in our VEN/AZA-treated AML cohort was 5.6%, in-line with incidence rates reported by recent similar studies. Furthermore, IFI incidence decreased as days from starting VEN/AZA therapy increased.

Keywords:  acute myeloid leukemia; azacitidine; invasive fungal infection; prophylaxis; venetoclax

DOI:  https://doi.org/10.1093/ofid/ofac486
STAR Protoc. 2022 Oct 13. pii: S2666-1667(22)00650-5. [Epub ahead of print]3(4): 101770

Multiomics data integration to reveal chromatin remodeling and reorganization induced by gene mutational synergy.

Haiyang Yun, Shabana Vohra, David Lara-Astiaso, Brian J P Huntly.

  Recurrent gene mutations often cooperate in a predefined stepwise and synergistic manner to alter global transcription, through directly or indirectly remodeling epigenetic landscape on linear and three-dimensional (3D) scales. Here, we present a multiomics data integration approach to investigate the impact of gene mutational synergy on transcription, chromatin states, and 3D chromatin organization in a murine leukemia model. This protocol provides an executable framework to study epigenetic remodeling induced by cooperating gene mutations and to identify the critical regulatory network involved. For complete details on the use and execution of this protocol, please refer to Yun et al. (2021).

Keywords:  Bioinformatics; Cancer; Genomics

DOI:  https://doi.org/10.1016/j.xpro.2022.101770
J Am Coll Cardiol. 2022 Oct 18. pii: S0735-1097(22)06564-0. [Epub ahead of print]80(16): 1545-1556

Impact of Clonal Hematopoiesis in Patients With Cardiogenic Shock Complicating Acute Myocardial Infarction.

Matthias Böhme, Steffen Desch, Maciej Rosolowski, Markus Scholz, Knut Krohn, Petra Büttner, Michael Cross, Janine Kirchberg, Karl-Philipp Rommel, Janine Pöss, Anne Freund, Ronny Baber, Berend Isermann, Uta Ceglarek, Klaus H Metzeler, Uwe Platzbecker, Holger Thiele.

   BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) is common in elderly individuals and is associated with an increased risk of both hematologic malignancies and cardiovascular disease. The impact of CHIP on the outcomes for patients with cardiogenic shock (CS) complicating acute myocardial infarction (AMI) remains undetermined.
OBJECTIVES: The purpose of this study was to determine the prognostic impact of CHIP in CS after AMI.
METHODS: Blood samples were obtained at randomization from 446 patients included in the CULPRIT-SHOCK (Culprit Lesion Only vs Multivessel Percutaneous Coronary Intervention in Cardiogenic Shock; NCT01927549) trial. CHIP was assessed using a next-generation sequencing approach targeting the most commonly mutated genes; the primary outcome at 30 days comprised all-cause mortality and renal replacement therapy.
RESULTS: CHIP variants at ≥2% variant allele frequency were detected in 29% (n = 129), most commonly in the DNMT3A or TET2 genes, which harbored 47% and 36% of all mutations, respectively. Compared to non-CHIP patients, CHIP carriers were older and had decreased renal function and increased levels of N-terminal pro-B-type natriuretic peptide and inflammatory biomarkers. CHIP carriers had worse short-term outcomes measured either as mortality or as the combined clinical endpoint of mortality or severe renal failure within 30 days. Association of CHIP with the combined endpoint was independent of age and biomarkers reflecting kidney function, heart failure severity, and inflammation (OR: 1.83; 95% CI: 1.05-3.21; P = 0.03) but not significant regarding all-cause mortality (OR: 1.67; 95% CI: 0.96-2.90; P = 0.069).
CONCLUSIONS: CHIP is frequent among AMI and CS patients and is associated with impaired clinical outcome. CHIP assessment may facilitate risk stratification in patients with CS and imply novel treatment targets. (Culprit Lesion Only vs Multivessel Percutaneous Coronary Intervention in Cardiogenic Shock [CULPRIT-SHOCK]; NCT01927549).

Keywords:  acute myocardial infarction; cardiogenic shock; clonal hematopoiesis of indeterminate potential; percutaneous coronary intervention

DOI:  https://doi.org/10.1016/j.jacc.2022.08.740
Br J Haematol. 2022 Oct 10.

Dasatinib plus Peg-Interferon alpha 2b combination in newly diagnosed chronic phase chronic myeloid leukaemia: Results of a multicenter phase 2 study (DASA-PegIFN study).

French CML Group (FiLMC)

  Superior rates of deep molecular response (DMR) have been reported with the combination of tyrosine kinase inhibitors and pegylated-interferon-alpha (Peg-IFN) in patients with newly diagnosed chronic phase-chronic myeloid leukaemia (CP-CML). In this setting, this study investigated the efficacy and safety of dasatinib combined to Peg-IFN-α2b (Dasa-PegIFN, NCT01872442). A total of 79 patients (age ≤65 years) started dasatinib; 61 were eligible for Peg-IFNα-2b add-on therapy at month 3 for a maximum 21-months duration. Dasatinib was continued thereafter. The primary endpoint was the cumulative rate of molecular response 4.5 log (MR4.5 ) by 12 months. The results are reported for the 5-year duration of the study. Grade 3 neutropenia was frequent with the combination but did not induce severe infection (one of grade 3). Other adverse events were generally low grade (4% of grade 3-4) and expected. Seventy-nine per cent and 61% of patients continued the Peg-IFN until months 12 and 24, respectively. Overall, at these time points, MR4.5 rates were 25% and 38%, respectively. Thereafter, 32% and 46% of patients achieved a sustained (≥2 years) MR4.5 or MR4 , respectively. This work established the feasibility and high rates of achievement of early and sustained DMR (a prerequisite for treatment-free-remission) with dasatinib and Peg-IFNα-2b combination as initial therapy.

Keywords:  chronic myeloid leukaemia; clinical trial; deep molecular response; pegylated-interferon; tyrosine kinase inhibitor

DOI:  https://doi.org/10.1111/bjh.18486
Proc Natl Acad Sci U S A. 2022 Oct 18. 119(42): e2213718119

LYL1 facilitates AETFC assembly and gene activation by recruiting CARM1 in t(8;21) AML.

Qian Chen, Murat A Cevher, Qi Jiang, Saisai Wang, Xiaojian Sun, Robert G Roeder, Mo Chen.

  Transcription factors (TFs) play critical roles in hematopoiesis, and their aberrant expression can lead to various types of leukemia. The t(8;21) leukemogenic fusion protein AML1-ETO (AE) is the most common fusion protein in acute myeloid leukemia and can enhance hematopoietic stem cell renewal while blocking differentiation. A key question in understanding AE-mediated leukemia is what determines the choice of AE to activate self-renewal genes or repress differentiation genes. Toward the resolution of this problem, we earlier showed that AE resides in the stable AETFC complex and that its components colocalize on up- or down-regulated target genes and are essential for leukemogenesis. In the current study, using biochemical and genomic approaches, we show that AE-containing complexes are heterogeneous, and that assembly of the larger AETFC (containing AE, CBFβ, HEB, E2A, LYL1, LMO2, and LDB1) requires LYL1. Furthermore, we provide strong evidence that the LYL1-containing AETFC preferentially binds to active enhancers and promotes AE-dependent gene activation. Moreover, we show that coactivator CARM1 interacts with AETFC and facilitates gene activation by AETFC. Collectively, this study describes a role of oncoprotein LYL1 in AETFC assembly and gene activation by recruiting CARM1 to chromatin for AML cell survival.

Keywords:  AML1–ETO; CARM1; E proteins; LYL1; acute myeloid leukemia

DOI:  https://doi.org/10.1073/pnas.2213718119
Blood Adv. 2022 Oct 11. pii: bloodadvances.2022007497. [Epub ahead of print]

Clonal hematopoiesis in patients with stem cell mobilization failure: a nested case-control study.

Carin Hazenberg, Aniek O de Graaf, René Mulder, Laura Bungener, Maaike G J M van Bergen, André B Mulder, Goda Choi, Jan Jacob Schuringa, Marco de Groot, Edo Vellenga, Joop H Jansen, Gerwin Huls, Isabelle A van Zeventer.

Inadequate mobilization of peripheral blood progenitor cells (PBPC) is a limiting factor to proceed with autologous hematopoietic cell transplantation (auto-HCT). To assess the impact of clonal hematopoiesis (CH) on mobilization failure of PBPC for auto-HCT, we investigated the characteristics of poor mobilizers (with total PBPC collection <2x106 CD34+ cells/kg) in a consecutive single center cohort of 776 patients. Targeted error corrected next-generation sequencing of 28 genes was performed in a nested case-control cohort of 90 poor mobilizers and 89 matched controls. CH was detected in 48 out of 179 patients (27%), with most patients carrying a single mutation. The presence of CH (detected at VAF ≥1%) did not associate with poor mobilization potential (31% versus 22% in controls, OR 1.55, 95%CI 0.76-3.23, P=0.238). PPM1D mutations were detected more often in poor mobilizers (P=0.005). In addition, TP53 mutations in this cohort were detected exclusively in patients with poor mobilization potential (P=0.06). The incidence of therapy-related myeloid neoplasms (t-MN) was higher among patients with mobilization failure (P=0.014). Although poor mobilizers experienced worse overall survival (P=0.019), this was not affected by the presence of CH. We conclude that CH at low VAF (1-10%) is common at time of stem cell mobilization. TP53 mutations and PPM1D mutations associate with poor mobilization potential and their role in subsequent development of t-MN in these individuals should be established.

DOI: https://doi.org/10.1182/bloodadvances.2022007497
Blood Adv. 2022 Oct 14. pii: bloodadvances.2022008572. [Epub ahead of print]

Analysis of Disparities in Time to Allogeneic Transplantation in Adults with Acute Myelogenous Leukemia.

Warren B Fingrut, Boglarka Gyurkocza, Jessica R Flynn, Eric Davis, Sean M Devlin, Andromachi Scaradavou, Stephanie Chinapen, Sean Quach, Christina Cho, Sergio A Giralt, Ann A Jakubowski, Richard J Lin, Esperanza B Papadopoulos, Miguel-Angel Perales, Doris M Ponce, Brian Shaffer, Roni Tamari, James W Young, Ioannis Politikos, Juliet N Barker.

While alternative donors extend transplant access, whether recipient ancestry impacts the time to allogeneic transplant is not established. We analyzed the likelihood of clinically significant delays to allograft by patient ancestry in 313 transplanted adult acute myelogenous leukemia (AML) patients. Non-European ancestry patients (n = 99) were more likely than Europeans (n = 214) to receive HLA-mismatched donor allografts (45% versus 24%, p < 0.001). Overall, the median time from transplant indication to allograft was 127 days (range 57-1,683). In multivariable analysis, non-Europeans had an increased risk of prolonged indication-transplant time > 180 days (OR 2.1, 95%CI:1.4-5.6, p = 0.012) due to significant delays in indication-consult > 90 days (OR 2.8, 95%CI:1.3-4.5, p = 0.005) and consult-transplant > 120 days (OR 2.4, 95%CI:1.2-3.7, p = 0.005). Compared to recipients of HLA-matched unrelated donors (URD), HLA-mismatched adult donor recipients were at increased risk of delayed indication-transplant whereas matched sibling and cord blood recipients were at lower risk. Sub-analysis showed more indication-transplant delays in non-European versus European 8/8 URD recipients (44% versus 19% > 180 days, p = 0.004). Finally, the pandemic further exacerbated delays for non-Europeans. In summary, while non-European AML patients are less likely to receive 8/8 URDs as expected, if they do their transplants are delayed. HLA-identical siblings and cord blood facilitate the fastest transplants regardless of patient ancestry whereas other adult donor transplants are delayed. Strategies to mitigate referral barriers, hasten donor evaluation, and utilize all alternative donor sources are critical to ensure timely transplantation for AML patients.

DOI: https://doi.org/10.1182/bloodadvances.2022008572
Leukemia. 2022 Oct 12.

Comparison of the revised 4th (2016) and 5th (2022) editions of the World Health Organization classification of myelodysplastic neoplasms.

Yudi Zhang, Junying Wu, Tiejun Qin, Zefeng Xu, Shiqiang Qu, Lijuan Pan, Bing Li, Huijun Wang, Peihong Zhang, Xin Yan, Jingye Gong, Qingyan Gao, Robert Peter Gale, Zhijian Xiao.

We used data from 852 consecutive subjects with myelodysplastic neoplasms (MDS) diagnosed according to the 2016 (revised 4th) World Health Organization (WHO) criteria to evaluate the 2022 (5th) edition WHO classification of MDS. 30 subjects previously classified as MDS with an NPM1 mutation were re-classified as acute myeloid leukaemia (AML). 9 subjects previously classified as MDS-U were re-classified to clonal cytopenia of undetermined significance (CCUS). The remaining 813 subjects were diagnosed as: MDS-5q (N = 11 [1%]), MDS-SF3B1 (N = 70 [9%]), MDS-biTP53 (N = 53 [7%]), MDS-LB (N = 293 [36%]), MDS-h (N = 80 [10%]), MDS-IB1 (N = 161 [20%]), MDS-IB2 (N = 103 [13%]) and MDS-f (N = 42 [5%]) and MDS-biTP53 (N = 53 [7%]). 34 of these subjects came from the 53 (64%) MDS-biTP53 previously diagnosed as MDS-EB. Median survival of subjects classified as MDS using the WHO 2022 criteria was 45 months (95% Confidence Interval [CI], 34, 56 months). Subjects re-classified as MDS-biTP53 and MDS-f had significantly briefer median survivals compared with other MDS sub-types (10 months, [8, 12 months] and 15 months [8, 23 months]). In conclusion, our analyses support the refinements made in the WHO 2022 proposal.

DOI: https://doi.org/10.1038/s41375-022-01718-7
Lancet Haematol. 2022 Oct 07. pii: S2352-3026(22)00265-4. [Epub ahead of print]

Current status of phase 3 clinical trials in high-risk myelodysplastic syndromes: pitfalls and recommendations.

Guillermo Garcia-Manero.

Single-agent hypomethylating agents remain the cornerstone of treatment for patients with high-risk myelodysplastic syndromes. Although these agents have clinical activity and can improve the overall survival of these patients, their impact on the natural history of myelodysplastic syndromes is only partial. Therefore, we need either newer agents or combinations that could have a greater impact on the survival of our patients. Over the past decade there has been an increased effort in drug development for myelodysplastic syndromes. Hypomethylating agent combinations that have been explored over the past decade include agents that block mutant TP53, NEDD inhibitors, BCL-2 inhibitors, and antibodies such as sabatolimab or magrolimab. Despite initial encouraging results, two registration trials from 2021 and 2022 have not been successful in improving outcomes when compared with single-agent hypomethylating agents. Here, I summarise the current status of ongoing phase 3 trials for patients with untreated high-risk myelodysplastic syndromes and provide some suggestions for future designs.

DOI: https://doi.org/10.1016/S2352-3026(22)00265-4
JCI Insight. 2022 Oct 11. pii: e163040. [Epub ahead of print]

Pathogenicity and impact of HLA class I alleles in aplastic anemia patients of different ethnicities.

Timothy S Olson, Benjamin F Frost, Jamie L Duke, Marian Dribus, Hongbo M Xie, Zachary D Prudowsky, Elissa Furutani, Jonas Gudera, Yash B Shah, Deborah Ferriola, Amalia Dinou, Ioanna Pagkrati, Soyoung Kim, Yixi Xu, Meilun He, Shannon Zheng, Sally Nijim, Ping Lin, Chong Xu, Taizo Nakano, Joseph H Oved, Beatriz M Carreno, Yung-Tsi Bolon, Shahinaz M Gadalla, Steven Ge Marsh, Sophie Paczesny, Stephanie J Lee, Dimitrios S Monos, Akiko Shimamura, Alison A Bertuch, Loren Gragert, Stephen Spellman, Daria V Babushok.

  Acquired aplastic anemia (AA) is caused by autoreactive T-cell-mediated destruction of early hematopoietic cells. Somatic loss of human leukocyte antigen (HLA) Class I alleles was identified as a mechanism of immune escape in surviving hematopoietic cells of some AA patients. However, pathogenicity, structural characteristics and clinical impact of specific HLA alleles in AA remain poorly understood. Here, we evaluated somatic HLA loss in 505 AA patients from two multi-institutional cohorts. Using a combination of HLA mutation frequencies, peptide-binding structures, and association with AA in an independent cohort of 6,323 patients from the National Marrow Donor Program, we identified 19 AA risk alleles and 12 non-risk alleles and established a novel AA HLA pathogenicity stratification. Our results define pathogenicity for the majority of common HLA-A/B alleles across diverse populations. Our study demonstrates that HLA alleles confer different risks of developing AA, but once AA develops, specific alleles are not associated with response to immunosuppression or trans-plant outcomes. However, higher pathogenicity alleles, particularly HLA-B*14:02, are associated with higher rates of clonal evolution in adult AA patients. Our study provides novel insights into the immune pathogenesis of AA, opening the door to future autoantigen identification and improved under-standing of clonal evolution in AA.

Keywords:  Autoimmune diseases; Autoimmunity; Clonal selection; Hematology; Hematopoietic stem cells

DOI:  https://doi.org/10.1172/jci.insight.163040
Leukemia. 2022 Oct 14.

DDX41 coordinates RNA splicing and transcriptional elongation to prevent DNA replication stress in hematopoietic cells.

Satoru Shinriki, Mayumi Hirayama, Akiko Nagamachi, Akihiko Yokoyama, Takeshi Kawamura, Akinori Kanai, Hidehiko Kawai, Junichi Iwakiri, Rin Liu, Manabu Maeshiro, Saruul Tungalag, Masayoshi Tasaki, Mitsuharu Ueda, Kazuhito Tomizawa, Naoyuki Kataoka, Takashi Ideue, Yutaka Suzuki, Kiyoshi Asai, Tokio Tani, Toshiya Inaba, Hirotaka Matsui.

Myeloid malignancies with DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5' splice site (5'SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5'SS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.

DOI: https://doi.org/10.1038/s41375-022-01708-9
Clin Cancer Res. 2022 Oct 12. pii: CCR-22-1810. [Epub ahead of print]

Safety, outcomes and T cell characteristics in patients with relapsed or refractory MDS or CMML treated with atezolizumab in combination with guadecitabine.

Casey L O'Connell, Maria R Baer, Andreas Due Ørskov, Sunil Kumar Saini, Vu H Duong, Patricia Kropf, Jakob Werner Hansen, Denice Tsao-Wei, Hyo Sik Jang, Ashkan Emadi, Staffan Holmberg-Thyden, Jack Cowland, Brett T Brinker, Kristin Horwood, Ryan Burgos, Galen Hostetter, Benjamin A Youngblood, Sine Reker Hadrup, Jean-Pierre Issa, Peter Jones, Stephen B Baylin, Imran Siddiqi, Kirsten Grønbæk.

PURPOSE: We hypothesized that resistance to hypomethylating agents (HMAs) among patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) would be overcome by combining a PD-L1 antibody with an HMA.
MATERIALS AND METHODS: We conducted a phase 1/2, multicenter clinical trial for patients with MDS not achieving an IWG-response after at least 4 cycles of an HMA "refractory" or progressing after a response "relapsed" with 3+ or higher risk MDS by the revised International Prognostic Scoring System (IPSS-R) and CMML-1 or -2. Phase I consisted of a 3+3 dose escalation design beginning with guadecitabine at 30 mg/m2 and escalating to 60 mg/m2 days 1-5 with fixed-dose atezolizumab: 840mg IV days 8 and 22 of a 28-day cycle. Primary endpoints were safety and tolerability; secondary endpoints were overall response rate (ORR) and survival.
RESULTS: Thirty-three patients, median age 73 (range 54-85), were treated. Thirty patients had MDS and 3 had CMML, with 30% relapsed and 70% refractory. No DLTs were observed in Phase I. There were 3 (9%) deaths in ≤30 days. Five patients (16%) came off study for drug-related toxicity. Immune-related adverse events (IRAEs) occurred in 12 (36%) patients (4 grade 3, 3 grade 2, 5 grade1). ORR was 33% (95% CI: 19, 52%) with 2 complete remission (CR), 3 hematologic improvement (HI), 5 marrow CR, 1 partial remission (PR). Median overall survival (mOS) was 15.1 (95% CI: 8.5, 25.3) months.
CONCLUSION: Guadecitabine with atezolizumab has modest efficacy with manageable IRAEs and typical cytopenia-related safety concerns for patients with R/R MDS and CMML.

DOI: https://doi.org/10.1158/1078-0432.CCR-22-1810
STAR Protoc. 2022 Oct 12. pii: S2666-1667(22)00644-X. [Epub ahead of print]3(4): 101764

Isolation, culture, and immunophenotypic analysis of bone marrow HSPCs from patients with myelodysplastic syndromes.

Irene Ganan-Gomez, Karen Clise-Dwyer, Simona Colla.

  Drug testing assays in hematopoietic stem and progenitor cells (HSPCs) are fundamental in biological studies of myelodysplastic syndromes (MDS) but have historically entailed a technical challenge. This protocol allows the efficient isolation of MDS HSPCs from bone marrow mononuclear cell fractions and their culturing with the support of stromal cells for improved maintenance during drug testing. Lastly, specific steps are given to quantify surviving cells and assess changes in the HSPC hierarchies. For complete details on the use and execution of this protocol, please refer to Ganan-Gomez et al. (2022).

Keywords:  Cancer; Cell Biology; Cell culture; Cell isolation; Cell separation/fractionation; Flow Cytometry/Mass Cytometry; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2022.101764
Leuk Lymphoma. 2022 Oct 12. 1-8

Incidence of adverse effects in patients receiving ponatinib with concomitant azole antifungals.

Kayleigh R Marx, Caitlin R Rausch, Alexandra R Lovell, Nicholas J Short, Shilpa Paul, Nitin Jain, Jenessa Lee, J Michael Savoy, Farhad Ravandi, Elias Jabbour.

  Ponatinib plus Hyper-CVAD yields a five-year overall survival of 73% in patients with Philadelphia-positive acute lymphoblastic leukemia. Ponatinib dose intensity is associated with increased incidence of adverse effects (AEs), including vascular events. Ponatinib combined with azole antifungals may further increase the risk of AEs due to increased ponatinib exposure. We reviewed 53 patients who received ponatinib with intensive (n = 39; 74%) or low-intensity chemotherapy (n = 14; 26%). Forty-eight patients (91%) received concomitant azole. Ponatinib was commonly initiated at 30 mg (n = 30; 57%) or 45 mg daily (n = 21; 40%). Twenty-six patients (49%) experienced at least one grade ≥3 AE possibly related to ponatinib; 19 (73%) were receiving a ponatinib dose equivalent ≥30mg and 58% >45mg. Eight patients (15%) experienced 10 vascular events, including 1 arterial event; 9 occurred on a ponatinib dose equivalent ≥30mg and 5 occurred while on an azole. Vascular events pose a clinical challenge with the risk potentially increasing with concomitant azoles.

Keywords:  Ponatinib; ponatinib adverse events; ponatinib and azoles

DOI:  https://doi.org/10.1080/10428194.2022.2131409
Cell Death Dis. 2022 Oct 13. 13(10): 869

c-Mpl-del, a c-Mpl alternative splicing isoform, promotes AMKL progression and chemoresistance.

Fei Li, Yuanyan Xiong, Mo Yang, Peiling Chen, Jingkai Zhang, Qiong Wang, Miao Xu, Yiming Wang, Zuyong He, Xin Zhao, Junyu Huang, Xiaoqiong Gu, Li Zhang, Rui Sun, Xunsha Sun, Jingyao Li, Jinxin Ou, Ting Xu, Xueying Huang, Yange Cao, Xiaohong Ruby Xu, Danielle Karakas, June Li, Heyu Ni, Qing Zhang.

Acute megakaryocytic leukemia (AMKL) is a clinically heterogeneous subtype of acute myeloid leukemia characterized by unrestricted megakaryoblast proliferation and poor prognosis. Thrombopoietin receptor c-Mpl is a primary regulator of megakaryopoeisis and a potent mitogenic receptor. Aberrant c-Mpl signaling has been implicated in a myriad of myeloid proliferative disorders, some of which can lead to AMKL, however, the role of c-Mpl in AMKL progression remains largely unexplored. Here, we identified increased expression of a c-Mpl alternative splicing isoform, c-Mpl-del, in AMKL patients. We found that c-Mpl-del expression was associated with enhanced AMKL cell proliferation and chemoresistance, and decreased survival in xenografted mice, while c-Mpl-del knockdown attenuated proliferation and restored apoptosis. Interestingly, we observed that c-Mpl-del exhibits preferential utilization of phosphorylated c-Mpl-del C-terminus Y607 and biased activation of PI3K/AKT pathway, which culminated in upregulation of GATA1 and downregulation of DDIT3-related apoptotic responses conducive to AMKL chemoresistance and proliferation. Thus, this study elucidates the critical roles of c-Mpl alternative splicing in AMKL progression and drug resistance, which may have important diagnostic and therapeutic implications for leukemia accelerated by c-Mpl-del overexpression.

DOI: https://doi.org/10.1038/s41419-022-05315-5
Blood. 2022 Oct 11. pii: blood.2022017561. [Epub ahead of print]

RUNX1-deficient human megakaryocytes demonstrate thrombopoietic, and platelet half-life and functional defects.

Kiwon Lee, Hyunsook Ahn, Brian Estevez, Mortimer Poncz.

Heterozygous defects in runt-related transcription factor-1 (RUNX1) are causative of a familial platelet disorder with associated myeloid malignancy (FPDMM). Since RUNX1-deficient animal models do not mimic FPDMM's bleeding disorder or leukemic risk, establishment of a proper model system is critical to understand the underlying mechanisms of the observed phenotype and to identify therapeutic interventions. We previously reported an in vitro-megakaryopoiesis system using human CD34+-hematopoietic stem and progenitor cells that recapitulated the FPDMM quantitative megakaryocyte defect by decreasing RUNX1 expression using a lentiviral short-hairpin RNA (shRNA for RUNX1 or shRX) strategy. We now show that shRX-megakaryocytes have a marked reduction in agonist responsiveness. We then infused shRX-megakaryocytes into immunocompromised NOD-SCID gamma (NSG) mice and demonstrated that these megakaryocytes released fewer platelets than megakaryocytes transfected with a non-targeting shRNA, and these platelets had a diminished half-life. The platelets were also poorly responsive to agonists, unable to correct thrombus formation in NSG mice homozygous for a R1326H mutation in von Willebrand Factor (VWFR1326H), which switches species-binding specificity of the VWF from mouse to human glycoprotein Iba. A small-molecule inhibitor RepSox, which blocks the transforming-growth factor beta pathway, and which rescued defective megakaryopoiesis in vitro, corrected the thrombopoietic defect, platelet half-life and agonist response, and thrombus formation in NSG/ VWFR1326H mice. Thus, this model recapitulates the defect in FPDMM megakaryocytes and platelets, identifies previously unrecognized defects in thrombopoiesis and platelet half-life, and demonstrates, for the first time, reversal of RUNX1 deficiency's hemostatic defects by a drug.

DOI: https://doi.org/10.1182/blood.2022017561
Ann Hematol. 2022 Oct 12.

The new diagnostic criteria for myelodysplasia-related acute myeloid leukemia is useful for predicting clinical outcome: comparison of the 4th and 5th World Health Organization classifications.

Hee Sue Park, Hee Kyung Kim, Hong-Sik Kim, Yaewon Yang, Hye Sook Han, Ki Hyeong Lee, Bo Ra Son, Jihyun Kwon.

  Mutations in myelodysplasia-related (MR) genes, rather than morphological features, have been included in the diagnostic criteria of the new 5th World Health Organization (WHO) classification for myelodysplastic syndrome (MDS)-associated acute myeloid leukemia (AML). This study compares the clinical relevance of the new criteria with those of the previous version. In a cohort of 135 patients with newly diagnosed AML, the MDS-related AML patients were classified according to the 5th and 4th edition of the WHO classification (AML, myelodysplasia-related [AML-MR5th] and AML with myelodysplasia-related changes [AML-MRC4th], respectively). The median age of the patients was 70.4 years. MR gene mutations were found in 48 patients (35.6%). Sixty-one patients (46.6%) were diagnosed with AML-MRC4th, while 71 patients (53.0%) were diagnosed with AML-MR5th. Patients with AML-MR5th were significantly older with significantly lower treatment response rate, higher recurrence rate, and shorter relapse-free survival after chemotherapy, whereas AML-MRC4th patients did not show any association with the treatment outcome. Overall, the following prognostic factors for survival were identified: age over 75 years, antecedent MDS or MDS/myeloproliferative neoplasm, chromosome 5 or 7 abnormalities, and KRAS and ZSZR2 mutations. The 5th WHO classification is more useful for predicting the treatment response of patients with AML-MR than the previous version. Among the MR genes, ZSZR2 mutations were found to be independent prognostic factors affecting survival.

Keywords:  Acute myeloid leukemia; Mutation; Myelodysplasia-related genes; World Health Organization classification

DOI:  https://doi.org/10.1007/s00277-022-05002-7
Bone Marrow Transplant. 2022 Oct 10.

Donor lymphocyte infusions after haploidentical stem cell transplantation with PTCY: A study on behalf of the EBMT cellular therapy & immunobiology working party.

Nicole Santoro, Jarl E Mooyaart, Raynier Devillier, Yener Koc, Jan Vydra, Luca Castagna, Zafer Gülbas, José Diez Martin, Mercedes Colorado Araujo, Alexander Kulagin, Mutlu Arat, Concepcion Herrera Arroyo, Maria Paola Martelli, Mauro Di Ianni, Jorinde D Hoogenboom, Liesbeth C de Wreede, Annalisa Ruggeri, Christian Chabannon.

Donor lymphocyte infusion (DLI) is a treatment option to prevent or treat relapse after allogeneic hematopoietic cell transplantation (HCT). We here report data for 173 patients who received one or multiple DLIs after haploidentical-HCT with post-transplant cyclophosphamide (PTCY) at 47 EBMT centers from 2009 to 2018. Indication for DLI was: prophylactic for 59(34.3%), preemptive for 20(11.6%), and therapeutic for 93(54.1%). For the prophylactic group, the median number of DLIs was 1 (IQR:1-2.5) with a median first dose of 0.1 × 106 CD3+ T cell/kg, for the preemptive 2 (IQR:1-3) with 0.5 × 106 CD3+ T cell/kg, for the therapeutic 1 (IQR:1-3) with 1 × 106CD3+ Tcell/kg, respectively. OS after first DLI was 61% (46-75%) for prophylactic, 40% (19-61%) for preemptive, and 22% (13-31%) for therapeutic. CI of II-IV aGVHD and cGVHD was 17%(7-27%) and 53% (40-67%) for the prophylactic, 20% (2-38%) and 21% (3-39%) for the preemptive, 17% (9-24%) and 24% (15-33%) for the therapeutic group, respectively. Our data show great variability in the indications and modalities of DLI across responding EBMT centers. Survival rates remain relatively low in patients with active disease. While the cumulative incidence of aGVHD appears acceptable, we showed a high incidence proportion of cGVHD in the prophylactic group, compared with preemptive and therapeutic DLI. These data should be investigated further in prospective clinical trials.

DOI: https://doi.org/10.1038/s41409-022-01839-8
Exp Hematol. 2022 Oct 09. pii: S0301-472X(22)00704-4. [Epub ahead of print]

HSC derived fatty acid oxidation in steady state and stressed haematopoiesis.

Jayna J Mistry, Kristian Bowles, Stuart A Rushworth.

  Metabolism impacts all cellular functions and plays a fundamental role in physiology. Metabolic regulation in hematopoiesis is dynamically regulated under steady state and stress conditions. It is clear, that hematopoietic stem cells (HSCs) impose different energy demands and flexibility during maintenance compared to stressed conditions. However, the cellular and molecular mechanism underlying the metabolic regulation in HSCs remains poorly understand. In this review we focus on defining the role of fatty acid oxidation (FAO) in HSC. We will first review the existing literature describing FAO in HSCs under steady state hematopoiesis. Next, we will describe the models used to examine HSCs under stress conditions and finally, we will describe how infection causes a shift towards FAO in HSC and the impact of using this pathway has on emergency hematopoiesis.

Keywords:  Mitochondria; Stem cell; b-oxidation; metabolism

DOI:  https://doi.org/10.1016/j.exphem.2022.10.003
Sci Transl Med. 2022 Oct 12. 14(666): eabm6391

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

Ani Grigoryan, Dimitra Zacharaki, Alexander Balhuizen, Christophe Rm Côme, Alejandro Garcia Garcia, David Hidalgo Gil, Anne-Katrine Frank, Kristina Aaltonen, Adriana Mañas, Javanshir Esfandyari, Pontus Kjellman, Emelie Englund, Carmen Rodriguez, Wondossen Sime, Ramin Massoumi, Nasim Kalantari, Sujeethkumar Prithiviraj, Yuan Li, Steven J Dupard, Hanna Isaksson, Chris D Madsen, Bo T Porse, Daniel Bexell, Paul E Bourgine.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

DOI: https://doi.org/10.1126/scitranslmed.abm6391