bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2020‒08‒23
twenty papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Cell Stem Cell. 2020 Aug 14. pii: S1934-5909(20)30359-3. [Epub ahead of print]
    Jones CL, Stevens BM, Pollyea DA, Culp-Hill R, Reisz JA, Nemkov T, Gehrke S, Gamboni F, Krug A, Winters A, Pei S, Gustafson A, Ye H, Inguva A, Amaya M, Minhajuddin M, Abbott D, Becker MW, DeGregori J, Smith CA, D'Alessandro A, Jordan CT.
      We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.
    Keywords:  NAD+; NAMPT; acute myeloid leukemia; leukemia stem cells; metabolism; nicotinamide; oxidative phosphorylation; relapse; therapy resistance; venetoclax
  2. Nat Commun. 2020 Aug 18. 11(1): 4147
    Rai S, Tanaka H, Suzuki M, Espinoza JL, Kumode T, Tanimura A, Yokota T, Oritani K, Watanabe T, Kanakura Y, Matsumura I.
      Mutated receptor tyrosine kinases (MT-RTKs) such as internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3 ITD) and a point mutation KIT D816V are driver mutations for acute myeloid leukemia (AML). Clathrin assembly lymphoid myeloid leukemia protein (CALM) regulates intracellular transport of RTKs, however, the precise role for MT-RTKs remains elusive. We here show that CALM knock down leads to severely impaired FLT3 ITD- or KIT D814V-dependent cell growth compared to marginal influence on wild-type FLT3- or KIT-mediated cell growth. An antipsychotic drug chlorpromazine (CPZ) suppresses the growth of primary AML samples, and human CD34+CD38- AML cells including AML initiating cells with MT-RTKs in vitro and in vivo. Mechanistically, CPZ reduces CALM protein at post transcriptional level and perturbs the intracellular localization of MT-RTKs, thereby blocking their signaling. Our study presents a therapeutic strategy for AML with MT-RTKs by altering the intracellular localization of MT-RTKs using CPZ.
  3. Cancer Discov. 2020 Aug 21. pii: CD-19-0970. [Epub ahead of print]
    Su A, Ling F, Vaganay C, Sodaro G, Benaksas C, Dal Bello R, Forget A, Pardieu B, Lin KH, Rutter JC, Bassil CF, Fortin G, Pasanisi J, Antony-Debre I, Alexe G, Benoist JF, Pruvost A, Pikman Y, Qi J, Schlageter MH, Micol JB, Roti G, Cluzeau T, Dombret H, Preudhomme C, Fenouille N, Benajiba L, Golan HM, Stegmaier K, Lobry C, Wood KC, Itzykson R, Puissant A.
      Deciphering the impact of metabolic intervention on response to anticancer therapy may elucidate a path toward improved clinical responses. Here, we identify amino acid-related pathways connected to the folate cycle whose activation predicts sensitivity to MYC-targeting therapies in acute myeloid leukemia (AML). We establish that folate restriction and deficiency of the rate-limiting folate cycle enzyme, MTHFR - which exhibits reduced-function polymorphisms in about 10% of Caucasians - induce resistance to MYC targeting by BET and CDK7 inhibitors in cell lines, primary patient samples, and syngeneic mouse models of AML. Further, this effect is abrogated by supplementation with the MTHFR enzymatic product, CH3-THF. Mechanistically, folate cycle disturbance reduces H3K27/K9 histone methylation and activates a SPI1 transcriptional program counteracting the effect of BET inhibition. Our data provide a rationale for screening MTHFR polymorphisms and the folate cycle status to nominate patients most likely to benefit from MYC-targeting therapies.
  4. Korean J Intern Med. 2020 Aug 20.
    Han H, Byun JM, Shin DY, Yoon SS, Koh Y, Hong J, Kim I, Lee C, Yoo H, Yun H, Kim MJ, Cho SI, Seong MW, Park SS.
      Background/Aims: Understanding leukemic stem cell (LSC) is important for acute myeloid leukemia (AML) treatment. However, association of LSC with patient prognosis and genetic information in AML patients is unclear.Methods: Here we investigated the associations between genetic information and the various LSC phenotypes, namely multipotent progenitor (MPP)-like, lymphoid primed multipotent progenitor (LMPP)-like and granulocyte-macrophage progenitors (GMP)-like LSC in 52 AML patients.
    Results: In secondary AML patients, MPP-like LSC was significantly higher than de novo AML (p = 0.0037). The proportion of MPP-like LSC was especially high in post-myeloproliferative neoplasm AML (p = 0.0485). There was no correlation between age and LSC phenotype. Mutations of KRAS and NRAS were observed in MPP-like LSC dominant patients, TP53 and ASXL1 mutations in LMPP-like LSC dominant patients, and CEBPA, DNMT3A and IDH1 mutations in GMP-like LSC dominant patients. Furthermore, KRAS mutation was significantly associated with MPP-like LSC expression (p = 0.0540), and TP53 mutation with LMPP-like LSC expression (p = 0.0276). When the patients were separated according to the combined risk including next generation sequencing data, the poorer the prognosis, the higher the LMPP-like LSC expression (p = 0.0052). This suggests that the dominant phenotype of LSC is one of the important factors in predicting the prognosis and treatment of AML.
    Conclusions: LSC phenotype in AML is closely associated with the recurrent mutations which has prognostic implication. Further research to confirm the meaning of LSC phenotype in the context of genetic aberration is warranted.
    Keywords:  Acute myeloid leukemia; Leukemic stem cell; Next generation sequencing; Prognosis
  5. Hum Pathol. 2020 Aug 13. pii: S0046-8177(20)30150-7. [Epub ahead of print]
    Jiang G, Capo-Chichi JM, Liu A, Atenafu EG, Guo R, Tierens A, Minden MD, Chang H.
      Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) generally confers poor prognosis; however, clinical outcome remains heterogeneous. We sought to further stratify this sub-entity of AML by performing a retrospective analysis of 179 adult AML-MRC patients diagnosed at our institution. Based on 2016 WHO diagnostic criteria, 44 (25%) had multilineage dysplasia alone (AML-MRC-M), 74 (41%) had history of MDS or MDS/MPN (AML-MRC-H), and 61 (34%) had MDS-related cytogenetics (AML-MRC-C). AML-MRC-M and hematopoietic stem cell transplantation (HSCT) were associated with prolonged EFS (P=0.0051 and P<0.0001, respectively) and OS (P=0.0015 and P<0.0001, respectively) while AML-MRC-C and age ≥60 years were associated with shorter EFS (P=0.028 and P=0.015, respectively) and OS (P=0.021 and P=0.013, respectively). Of note, NPM1mut did not affect patient outcome. Multivariable analysis confirmed HSCT and AML-MRC-C as independent predictors for EFS (P<0.0001 and P=0.0342, respectively) and OS (P<0.0001 and P=0.0295, respectively). AML-MRC-M was an independent predictor for OS (P=0.0449). When compared to a control group of 105 AML not otherwise specified with normal karyotype (NK-AML-NOS), AML-MRC-M patients had similar EFS and OS (P=0.99 and P=0.91, respectively). However, AML-MRC-H and AML-MRC-C were associated with shorter EFS and OS (P=0.0002 and P<0.0001, respectively) when compared to the same control group. In a subset of patients, NGS analysis showed AML-MRC-M was associated with ASXL1 mutation compared to NK-AML (56% vs 6%). In conclusion, AML-MRC-M demonstrates superior clinical outcome compared to the rest of AML-MRC. They have comparable outcome to NK-AML-NOS and this data suggests AML-MRC-M may be considered not to be classified in the same group as other AML-MRC patients.
    Keywords:  ASXL1 mutation; NPM1 mutation; acute myeloid leukemia; clinical outcome; multilineage dysplasia; myelodysplasia related changes
  6. Clin Cancer Res. 2020 Aug 14. pii: clincanres.2272.2020. [Epub ahead of print]
    Sill H, Zebisch A, Haase D.
      The tumor suppressor p53 exerts pivotal roles in hematopoietic stem cell (HSC) homeostasis. Mutations of the TP53 gene have recently been described in individuals with clonal hematopoieis conferring substantial risk of developing blood cancers. In patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), TP53 aberrations - mutations, deletions and a combination thereof - are encountered at a constant frequency of approximately 10%. These aberrations affect HSCs transforming them into preleukemic stem cells, pinpointing their central role in leukemogenesis. AML and MDS with TP53 aberrations are characterized by complex chromosomal aberrations. Respective patients experience a dismal long-term outcome following treatment with both, intensive and non-intensive regimens including novel agents like venetoclax combinations or even allogeneic hematopoietic stem cell transplantation. However, according to the 2016 WHO classification, AML and MDS with TP53 aberrations are still regarded separate disese entities. Based on their common biological and clinical features, we propose to classify AML and MDS with TP53 aberrations as a single, distinct stem cell disorder with a unique genetic make-up, comparable to the WHO classification of "AML with recurrent genetic abnormalities". This approach will have implications for basic and translational research endeavours, aid in harmonization of current treatment strategies and facilitate the development of master trials targeting a common deleterious driver event.
  7. Blood. 2020 Aug 18. pii: blood.2020005998. [Epub ahead of print]
    Kapp-Schwoerer S, Weber D, Corbacioglu A, Gaidzik VI, Paschka P, Krönke J, Theis F, Rücker FG, Teleanu MV, Panina E, Jahn N, Herzig JK, Kubanek L, Schrade A, Gohring G, Fiedler W, Kindler T, Schroeder T, Mayer K, Lübbert M, Wattad M, Götze K, Horst HA, Koller E, Wulf GG, Schleicher J, Bentz M, Krauter J, Bullinger L, Krzykalla J, Benner A, Schlenk RF, Thol F, Heuser M, Ganser A, Döhner H, Döhner K.
      Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1 mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TL) by RQ-PCR and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TL and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase III AMLSG 09-09 trial. 3733 bone marrow (BM) and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after two treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity consistently revealed as poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TL were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly higher proportion of patients achieving MRD negativity at EOT (56% vs 41%; P=.01). The betterreduction of NPM1mut TL after two treatment cycles in MRD-positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR 29.3% vs 45.7%, P=.009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction of NPM1mut TL across all treatment cycles leading to a significantly lower relapse rate. The AMLSG 09-09 trial was registered at as #NCT00893399.
  8. Leuk Lymphoma. 2020 Aug 19. 1-8
    Roboz GJ, Strickland SA, Litzow MR, Dalovisio A, Perl AE, Bonifacio G, Haines K, Barbera A, Purkayastha D, Sweet K.
      Approval of midostaurin, a multikinase inhibitor, in combination with chemotherapy for the treatment of adults with newly diagnosed FLT3 mutation-positive acute myeloid leukemia, was based on the phase 3 RATIFY trial results. RADIUS-X (NCT02624570) was an expanded access program providing access to midostaurin during regulatory review and extending the understanding of the safety and tolerability of midostaurin. Patients aged ≥18 years received midostaurin with 1-2 cycles of induction therapy (cytarabine plus daunorubicin or idarubicin) and ≤4 cycles of high-dose cytarabine consolidation chemotherapy or as single-agent maintenance therapy. The study enrolled 103 patients. No new safety events were observed; toxicities were not influenced by age, anthracycline choice, or coadministration of CYP3A4 inhibitors. The most common adverse events (AEs) were febrile neutropenia, nausea, and diarrhea. During maintenance, 46% of patients reported AEs. Midostaurin demonstrated a manageable safety profile and was associated with high transplant and low on-treatment relapse rates.
    Keywords:  Acute myeloid leukemia; FLT3; idarubicin; maintenance; midostaurin
  9. Blood Adv. 2020 Aug 25. 4(16): 3864-3874
    Grimm J, Jentzsch M, Bill M, Goldmann K, Schulz J, Niederwieser D, Platzbecker U, Schwind S.
      In 2017, an updated European LeukemiaNet (ELN) risk classification was published allocating patients with acute myeloid leukemia (AML) to 3 risk groups on the basis of certain cytogenetic and molecular aberrations. To date, studies of the prognostic significance of the ELN2017 risk classification in the context of an allogeneic hematopoietic stem cell transplantation (HSCT) are lacking. We performed risk stratification according to the ELN2017 classification in 234 patients with AML who underwent allogeneic HSCT as a consolidation therapy. In our cohort, the risk of 39.7% of the patients was classified as favorable, that of 12.8% as intermediate, and that of 47.4% as adverse. In the context of allogeneic HSCT, the assignment to the 3 ELN2017 risk groups retained its prognostic significance, with patients with favorable risk having the best prognosis and those with adverse risk having the worst one. Subgroup analyses showed that patients with a monosomal karyotype or TP53 mutation had considerably increased relapse rates, even in the adverse-risk group. When we analyzed the impact of digital droplet PCR-based measurable residual disease (MRD) before allogeneic HSCT, MRD+ patients had impaired prognoses, with cumulative incidence of relapse and overall survival comparable to those of patients classified as having an ELN2017 adverse genetic risk. This study is the first to demonstrate that the ELN2017 classification distinguishes the 3 risk groups with significantly distinct prognoses, even after allogeneic HSCT, and emphasizes the dismal prognosis of patients with AML with TP53 mutations, monosomal karyotype, or MRD positivity after allogeneic HSCT.
  10. Immunol Invest. 2020 Aug 20. 1-11
    Jin Z, Lan T, Zhao Y, Du J, Chen J, Lai J, Xu L, Chen S, Zhong X, Wu X, Li Y.
      The diverse structural and functional heterogeneity of γδ T cells is related to their distinct role in cancer immunity. The different phenotypes of γδ T cells in patients with acute myeloid leukemia (AML) is far from clear. In particular, the expression pattern of co-inhibitory and co-stimulatory receptors on γδ T cells remains unknown. In this study, we analyzed the distribution of γδ T cell subsets by expression of the immune checkpoint co-inhibitor TIGIT (T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain) and its competing co-stimulatory receptor CD226 in AML patients of different clinical statuses (including de novo AML, AML in non-remission (NR), and AML in complete remission (CR)). Our data demonstrated an imbalanced distribution of TIGIT and CD226 on γδ T cells with a decrease in CD226+ γδ T cells and an increase in TIGIT+ γδ T cells in de novo AML patients, while TIGIT-CD226+ γδ T cells were restored in AML patients who achieved CR after chemotherapy. Moreover, the patients who had higher TIGIT+CD226- γδ T cells showed lower overall survival rate for non-M3 AML, which may be considered a novel prognostic immune biomarker. In conclusion, our study reveals for the first time that imbalance in the TIGIT/CD226 axis might be related to different clinical outcomes for AML patients.ABBREVIATIONS: AML: acute myeloid leukemia; CR: complete remission; ICs: immune checkpoints; PD-1: programmed death-1; γδ T cells: gamma delta T cells; TCR: T cell receptor; MHC: major histocompatibility complex; TIGIT: T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain; NK: natural killer; PB: Peripheral blood; NR: non-remission; FAB: French-American-British; WHO: World Health Organization; HIs: healthy individuals; OS: overall survival.
    Keywords:  Acute myeloid leukemia; CD226; TIGIT; γδ T cells
  11. Biomark Res. 2020 ;8 30
    Guo Y, Sun H, Zhang D, Zhao Y, Shi M, Yang M, Xing S, Fu X, Bin T, Lu B, Wu S, Xu X, Xu X, Chen Y, Zhao ZJ.
      Background: Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML.Methods and results: In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods.
    Conclusions: We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.
    Keywords:  Acute myeloid leukemia; Detection of mutations; FLT3-TKD; Tyrosine kinase
  12. Expert Opin Pharmacother. 2020 Aug 18. 1-9
    Donker ML, Ossenkoppele GJ.
      INTRODUCTION: Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, but the results for patients with AML are still unsatisfactory. The discovery of new mutations in AML, including IDH mutations, has opened the door for treatment with targeted agents. Ivosidenib is a selective, potent inhibitor of the IDH1 mutant protein.AREAS COVERED: This review summarizes the mechanism of action, safety profile and efficacy of ivosidenib for patients with IDH1-mutated AML. The authors then provide their expert perspectives on the use of the drug including their future perspectives.
    EXPERT OPINION: Ivosidenib is a promising, most probably practice changing, new drug for the treatment of IDH1-mutated AML. Current phase III trials are ongoing to evaluate the addition of ivosidenib to the current standards-of-care. In the near future, more drug combinations are awaited. Challenges for the future include the development of resistance and establishing the duration of maintenance therapy.
    Keywords:  AML; Acute myeloid leukemia; IDH1; hematology; ivosidenib
  13. Clin Cancer Res. 2020 Aug 20. pii: clincanres.1064.2020. [Epub ahead of print]
    Walker A, Byrd JC, Blachly JS, Bhatnagar B, Mims A, Orwick S, Lin TL, Crosswell HE, Zhang D, Minden MD, Munugalavadla V, Long L, Liu J, Pan Y, Oellerich T, Serve H, Rao AV, Blum WG.
      PURPOSE: Spleen tyrosine kinase (SYK) signaling is a proposed target in acute myeloid leukemia (AML). Sensitivity to SYK inhibition has been linked to HOXA9 and MEIS1 overexpression in preclinical studies. This trial evaluated the safety and efficacy of entospletinib, a selective inhibitor of SYK, in combination with chemotherapy in untreated AML.METHODS: This was an international multicenter phase 1b/2 study: entospletinib dose escalation (standard 3+3 design between 200 mg and 400 mg BID) + 7+3 (cytarabine + daunorubicin) in phase 1b, and entospletinib dose expansion (400 mg BID) + 7+3 in phase 2.
    RESULTS: Fifty-three patients (n=12 phase 1b, n=41 phase 2) with previously untreated de novo (n=39) or secondary (n=14) AML enrolled (58% male, median age 60 years). The composite complete response with entospletinib + 7+3 was 70%. Patients with baseline HOXA9 and MEIS1 expression higher than the median had improved overall survival compared to patients with below median HOXA9 and MEIS1 expression. Common adverse events were cytopenias, febrile neutropenia, and infection. There were no dose-limiting toxicities. Entospletinib-related skin rash and hyperbilirubinemia were also observed.
    CONCLUSION: Entosplentib with intensive chemotherapy was well tolerated in AML patients. Improved survival was observed in patients with HOXA9/MEIS1 overexpression, contrasting published data demonstrating poor survival in such patients. A randomized study will be necessary to determine whether entospletinib was a mediator this observation.
  14. Cancer Discov. 2020 Aug 21. pii: CD-20-0312. [Epub ahead of print]
    Berrien-Elliott MM, Cashen AF, Cubitt CC, Neal CC, Wong P, Wagner JA, Foster M, Schappe T, Desai S, McClain E, Becker-Hapak M, Foltz JA, Cooper ML, Jaeger N, Nonavinkere Srivatsan S, Gao F, Romee R, Abboud CN, Uy GL, Westervelt P, Jacoby MA, Pusic I, Stockerl-Goldstein KE, Schroeder MA, DiPersio J, Fehniger TA.
      NK cells are an emerging cancer cellular therapy and potent mediators of anti-tumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in acute myeloid leukemia (AML) patients. However, the dynamic changes in phenotype that occur after NK cell transfer that impact patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell-treated AML patients using mass cytometry. These data identify a unique in vivo differentiated ML NK cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally-induced checkpoint important for ML, but not conventional NK cell responses to cancer. The frequency of CD8a+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK cell therapeutics.
  15. Exp Hematol. 2020 Aug 13. pii: S0301-472X(20)30349-0. [Epub ahead of print]
    Lin H, Damen JE, Walasek MA, Szilvassy SJ, Turhan A, Louis SA, Eaves AC, Wognum AW.
      Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classical colony-forming unit (CFU) assay. Compared to the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster, simpler, with more flexible readout methods for selecting candidates for further drug development.
    Keywords:  CFU assay; drug screening; hematotoxicity; liquid culture; primary AML sample; primary CML sample
  16. Cold Spring Harb Perspect Med. 2020 Aug 17. pii: a035477. [Epub ahead of print]
    Stahl M, Epstein-Peterson ZD, Intlekofer AM.
      Leukemias and lymphomas acquire the capacity for unrestrained cell growth and proliferation in conjunction with loss of responsiveness to molecular programs that promote terminal differentiation. Malignant cells generate the building blocks required for rapid cell division through both increased acquisition of nutrients from the environment and reprogrammed intermediary metabolism to shunt these molecules into producing the protein, lipids, and nucleic acids that comprise cell biomass. These accelerated metabolic processes require energy in the form of ATP and reducing equivalents in the form of NADPH, which power biosynthetic reactions and buffer oxidative stress encountered by the metabolically active cancer cell. Cancer-associated metabolic alterations can also promote accumulation or depletion of specific metabolites that directly regulate cell fate and function, thereby coupling metabolic reprogramming to dedifferentiation and stemness. This review will focus on the mechanisms by which leukemia and lymphoma cells rewire cellular metabolism to support: (1) bioenergetics, (2) biomass accumulation, (3) redox balance, and (4) differentiation blockade. We will further highlight examples of how specific pathways of leukemia and lymphoma metabolism confer therapeutic vulnerabilities that can be targeted to inhibit growth or promote differentiation.
  17. Leuk Lymphoma. 2020 Aug 20. 1-11
    Rivera-Torres N, Banas K, Kmiec EB.
      Clustered regularly interspaced palindromic repeats (CRISPR) with the associated (Cas) nuclease complexes have democratized genetic engineering through their precision and ease-of-use. We have applied a variation of this technology, known as CRISPR-directed mutagenesis (CDM), to reconstruct genetic profiles within the FLT3 gene of AML patients. We took advantage of the versatility of CDM and built expression vectors that, in combination with a specifically designed donor DNA fragment, recapitulate simple and complex mutations within the FLT3 gene. We generate insertions and point mutations including combinations of these mutations originating from individual patient samples. We then analyze how these complex genetic profiles modulate transformation of Ba/F3 cells. Our results show that FLT3 expression plasmids bearing patient-specific single or multiple mutations recapitulate cellular transformation properties induced by FLT3 ITDs and modify their sensitivity or resistance in response to established AML drugs as a function of these complex mutations.
    Keywords:  AML; CRISPR-directed mutagenesis; FLT-3
  18. Nat Commun. 2020 Aug 17. 11(1): 4115
    La Sala G, Michiels C, Kükenshöner T, Brandstoetter T, Maurer B, Koide A, Lau K, Pojer F, Koide S, Sexl V, Dumoutier L, Hantschel O.
      The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms.
  19. Cancers (Basel). 2020 Aug 12. pii: E2255. [Epub ahead of print]12(8):
    Rautenberg C, Bergmann A, Germing U, Fischermanns C, Pechtel S, Kaivers J, Jäger P, Schuler E, Haas R, Kobbe G, Schroeder T.
      To provide long-term outcome data and predictors for response and survival, we retrospectively analyzed all 151 patients with relapse of myeloid neoplasms after allogeneic hematopoietic stem cell transplantation (allo-HSCT) who were uniformly treated with first-line azacitidine (Aza) salvage therapy at our center. Patients were treated for molecular (39%) or hematologic relapse (61%), with a median of 5 cycles of Aza and at least one donor lymphocyte infusion in 70% of patients. Overall response was 46%, with 41% achieving complete (CR) and 5% achieving partial remission. CR was achieved after a median of 4 cycles and lasted for a median of 11 months (range 0.9 to 120 months). With a median follow-up of 22 months (range: 1 to 122 months), the 2-year survival rate was 38% ± 9%, including 17 patients with ongoing remission for >5 years. Based on results from multivariate analyses, molecular relapse and time to relapse were integrated into a score, clearly dividing patients into 3 subgroups with CR rates of 71%, 39%, and 29%; and 2-year survival rates of 64%, 38%, and 27%, respectively. In the subgroup of MDS and secondary AML, receiving upfront transplantation was associated with superior response and survival, and therefore pretransplant strategy was integrated together with relapse type into a MDS-sAML-specific score. Overall, Aza enables meaningful responses and long-term survival, which is a predictable with a simple-to-use scoring system.
    Keywords:  AML; MDS; allogeneic stem cell transplantation; azacitidine; relapse
  20. Nat Genet. 2020 Aug 17.
    Kim H, Nguyen NP, Turner K, Wu S, Gujar AD, Luebeck J, Liu J, Deshpande V, Rajkumar U, Namburi S, Amin SB, Yi E, Menghi F, Schulte JH, Henssen AG, Chang HY, Beck CR, Mischel PS, Bafna V, Verhaak RGW.
      Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution1-3; however, its frequency and clinical impact are unclear. Using computational analysis of whole-genome sequencing data from 3,212 cancer patients, we show that ecDNA amplification frequently occurs in most cancer types but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA, and the most common recurrent oncogene amplifications arose on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number-matched linear DNA, coupled with enhanced chromatin accessibility, and more frequently resulted in transcript fusions. Patients whose cancers carried ecDNA had significantly shorter survival, even when controlled for tissue type, than patients whose cancers were not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.