bims-tremyl Biomed News
on Therapy resistance biology in myeloid leukemia
Issue of 2022‒02‒13
twenty-four papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

  1. Blood Adv. 2022 Feb 07. pii: bloodadvances.2021006489. [Epub ahead of print]
      The phase 3 ADMIRAL trial demonstrated the superiority of the FLT3 inhibitor, gilteritinib, to salvage chemotherapy (SC) in patients with FLT3-mutated relapsed or refractory (R/R) acute myeloid leukemia (AML). Baseline co-mutations, FLT3-internal tandem duplication (ITD) allelic ratio and length, and treatment-emergent mutations were analyzed in patients in the ADMIRAL trial. Baseline co-mutations were grouped into molecular risk (DNA methylation/hydroxymethylation, transcription, chromatin-spliceosome, receptor tyrosine kinase [RTK]-Ras signaling, TP53-aneuploidy) or gene categories (NPM1, DNMT3A, DNMT3A/NPM1,WT-1, IDH1, and IDH2). Across all but one molecular risk category (TP53-aneuploidy) and all co-mutated gene subgroups, higher pre-transplant response rates and longer overall survival (OS) were observed with gilteritinib versus SC. Patients with DNMT3A/NPM1 co-mutations who received gilteritinib had the most favorable outcomes of any molecular subgroup analyzed. Survival outcomes with gilteritinib were not adversely affected by FLT3-ITD allelic ratio, FLT3-ITD length, or multiple FLT3-ITD mutations. Among patients who relapsed on gilteritinib, Ras/MAPK pathway and FLT3 F691L gene mutations were the most common mutational events associated with treatment resistance. However, the occurrence of Ras/MAPK pathway gene mutations at baseline did not preclude a clinical benefit from gilteritinib. Acquisition of multiple Ras/MAPK pathway gene mutations at relapse suggests a high level of pathway reactivation is needed to overcome the gilteritinib treatment effect. These findings provide insight into the R/R AML molecular profile and the impact of FLT3 inhibitors on mutational evolution associated with treatment resistance and benefit of gilteritinib across a wide spectrum of molecular and genetic subgroups in FLT3-mutated R/R AML.
  2. Haematologica. 2022 Feb 10.
      Less than a third of acute myeloid leukemia (AML) patients are cured by chemotherapy and/or hematopoietic stem cell transplantation (HSCT), highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells (LSCs), the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To better target these drug-resistant primitive leukemic cells, we have designed a CD34/CD3 bispecific T-cell engager (BiTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BiTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the single-cell level. Additionally, the BiTE triggered efficient T-cell-mediated depletion of CD34+ HSCs from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BiTE had in vivo efficacy by depleting CD34+ blasts and LSCs without side effects. Taken together, these data demonstrate robust antitumor effects of the CD34-specific BiTE supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.
  3. Exp Hematol. 2022 Feb 02. pii: S0301-472X(22)00001-7. [Epub ahead of print]
      Next-generation sequencing (NGS) is an excellent methodology for measuring residual disease in acute myeloid leukemia and survey several sub-clones simultaneously. Little experience exists regarding interpretation of differential clonal responses to therapy. We hypothesize that differential clonal response could best be studied in patients with residual disease at the time of response evaluation. We performed targeted panel sequencing of paired diagnostic and first treatment evaluation samples in 69 patients with residual disease by morphology or measurable residual disease (MRD) level >0.02. Five patients displayed a rising clone at the time of evaluation. A representative case showed the rising clone present only in the putative healthy stem cells (CD45lowCD34+CD38-CD123-CD7-) and not in the putative leukemic stem cells (CD34+CD38-CD123+CD7+) cells, thus representing non-malignant clonal hematopoiesis. In contrast, 17/43 evaluable patients displayed a differential response in genes related to the leukemic clone. 26/43 patients displayed a clonal response that followed the overall treatment response. Patients with a differential response had a better event-free survival (EFS) as well as overall survival (OS) than those where the clonal response followed the overall response (log-rank test, EFS P=0.045, OS, P=0.050). This indicates that when following multiple leukemia-related clones the less chemotherapy-responsive clone could, in some cases, have lesser relapse potential, contrary to what is known when using standard mutation or fusion transcript-based disease surveillance. In conclusion, our results confirm the potential of refining MRD assessments by following multiple clones and warrants further studies into the precise interpretations of multi-clone NGS-MRD assays.
    Keywords:  AML; MRD; NGS; clonal evolution; partial remission
  4. Cancer Lett. 2022 Feb 03. pii: S0304-3835(22)00048-9. [Epub ahead of print] 215579
      Recent advances in functional genomics have paved the path toward improvement in disease modeling for hematologic malignancies such as acute myeloid leukemia (AML). Among these functional genomic approaches include genome-wide CRISPR screens, induced pluripotent stem (iPS) cell technology, viral- or vector-based approaches, and informatics-based approaches. As a prime example, induction of pluripotency in somatic cells has remarkable potential for understanding of disease systems and gaining insight into therapeutics. In the recent years, the molecular applications of this iPS technology have extended to multiple arenas within hematologic malignancies. In AML, the generation of iPS cells has informed our understanding of clonal evolution at the stem cell level. Human AML-iPS cells have been shown to procure leukemic phenotypes and functions and can chart clonal evolution of the entire disease. Furthermore, sequential CRISPR-based editing of human iPS cells can model clonal dynamics and can identify novel therapeutic targets. Our group has recently modeled clonal dynamics in AML by annotating copy number variation against variant allele frequency to infer the subclonal structure of AML. This focused review highlights functional genomic approaches in AML - namely how dynamic expression of genetic content within specific contexts in hematopoietic cells contributes to biological behavior and phenotypes of disease. We explore the use of these novel technologies towards molecular target validation for AML as of 2022.
    Keywords:  Acute myeloid leukemia; CRISPR; Induced pluripotency; Reprogramming; iPS cells
  5. Am J Cancer Res. 2022 ;12(1): 427-444
      Differentiation arrest represents a distinct hallmark of acute myeloid leukemia (AML). Identification of differentiation-induction agents that are effective across various subtypes remains an unmet challenge. GTP biosynthesis is elevated in several types of cancers, considered to support uncontrolled tumor growth. Here we report that GTP overload by supplementation of guanosine, the nucleoside precursor of GTP, poises AML cells for differentiation and growth inhibition. Transcriptome profiling of guanosine-treated AML cells reveals a myeloid differentiation pattern. Importantly, the treatment compromises leukemia progression in AML xenograft models. Mechanistically, GTP overproduction requires sequential metabolic conversions executed by the purine salvage biosynthesis pathway including the involvement of purine nucleoside phosphorylase (PNP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). Taken together, our study offers novel metabolic insights tethering GTP homeostasis to myeloid differentiation and provides an experimental basis for further clinical investigations of guanosine or guanine nucleotides in the treatment of AML patients.
    Keywords:  Acute myeloid leukemia; HPRT1; PNP; guanosine 5’-triphosphate; myeloid differentiation
  6. JCI Insight. 2022 Feb 08. pii: e153768. [Epub ahead of print]7(3):
      Mosaic loss of chromosome Y (mLOY) in blood cells is one of the most frequent chromosome alterations in adult males. It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. However, whether there is a causal relationship between mLOY and human diseases is unknown. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. We found that mLOY led to dramatically increased DNA damage in HSPCs. Interestingly, HSPCs with mLOY displayed significantly enhanced reconstitution capacity and gave rise to clonal hematopoiesis in vivo. mLOY, which is associated with AML1-ETO translocation and p53 defects in patients with acute myeloid leukemia (AML), promoted AML in mice. Mechanistically, loss of KDM5D, a chromosome Y-specific histone 3 lysine 4 demethylase in both humans and mice, partially recapitulated mLOY in DNA damage and leukemogenesis. Thus, our study validates mLOY as a functional driver for clonal hematopoiesis and leukemogenesis.
    Keywords:  Genetic instability; Genetics; Hematology; Leukemias
  7. Blood Adv. 2022 Feb 07. pii: bloodadvances.2021006195. [Epub ahead of print]
      Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms.
  8. J Med Chem. 2022 Feb 11.
      Internal tandem duplication in the FLT3 receptor tyrosine kinase (FLT3/ITD mutation) occurs in approximately 25% of acute myeloid leukemia (AML) patients. To specifically target this driver mutation in AML, we assessed and compared the cell-based cytotoxicity of a diversity library (10,000 compounds) against the normal cell line BaF3 and the isogenic leukemic cell line BaF3/ITD. A benzoimidazole scaffold-based compound (HP1142) was identified as the most selective compound against a series of murine and human leukemia cells with FLT3/ITD. Novel benzoimidazole compounds were further designed to improve the aqueous solubility of HP1142. The most potent compound, HP1328, demonstrated desirable pharmaceutical and pharmacokinetic properties. Treatment with HP1328 significantly reduced the leukemia burden and prolonged the survival of mice with FLT3/ITD leukemia. Our findings establish the specific activity of the benzoimidazole compound against FLT3/ITD leukemia and warrant further investigation in this subset of leukemia patients with poor prognosis.
  9. Cent Eur J Immunol. 2021 ;46(4): 524-530
      Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by the presence of cytopenias, ineffective hematopoiesis and frequent transformation into secondary acute myeloid leukemia (secAML). Recent genomic studies provide unprecedented insight into the molecular landscape of clonal proliferation in MDS. Genetic diversity of both MDS and secAML subclones cannot be defined by a single somatic mutation. Mutations of the founding clone may survive over implemented chemotherapy and allogenic hematopoietic cell transplantation (alloHCT), but new subclonal mutations may also appear. Next generation sequencing (NGS) makes it possible to define the mutational profile of disease subclones during the treatment course and has a potential in pre- and post-alloHCT monitoring. Understanding the molecular pathophysiology of MDS may soon allow for monitoring the course of disease and personalized treatment depending on the mutational landscape. In the present paper we report, for the first time in MDS, ASXL1 c.1945G>T, TET2 c.4044+2dupT and c.4076G>T sequence variants. Moreover, we detected RUNX1 c.509-2A>C and SF3B1 c.1874G>T sequence variants. Furthermore, we verify the clinical utility of NGS and pyrosequencing in MDS and secAML.
    Keywords:  DNA sequence variants/mutations; NGS; allogenic hematopoietic cell transplantation; myelodysplastic syndrome; secondary acute myeloid leukemia
  10. Cancer Lett. 2022 Feb 02. pii: S0304-3835(22)00051-9. [Epub ahead of print]532 215582
      Interaction between stromal cells and acute myeloid leukemia (AML) cells in bone marrow (BM) is known to contribute importantly to chemoresistance and disease recurrence. Therefore, disruption of a crosstalk between AML cells and BM microenvironment may offer a promising therapeutic strategy for AML treatment. Here, we demonstrate that in a niche-like co-culture system, AML cells took up functional mitochondria from bone marrow stromal cells (BMSCs) and inhibition of such mitochondrial transfer by metformin, the most commonly prescribed drug for type 2 diabetes mellitus, significantly enhanced the chemosensitivity of AML cells co-cultured with BMSCs. The chemo-sensitizing effect of metformin was acted through reducing the mitochondrial transfer and mitochondrial oxidative phosphorylation (OXPHOS) in the recipient AML cells. In addition, metformin potentiated the antitumor efficacy of cytarabine (Ara-C) in vivo in an NCG immunodeficient mouse xenograft model by inhibiting the mitochondrial transfer and OXPHOS activity in the engrafted human AML cells. Altogether, this study identifies a potential application of metformin in sensitizing AML cells to chemotherapy and unveils a novel mechanism by which metformin executes such effect via blocking the mitochondrial transfer from stromal cells to AML cells.
    Keywords:  Chemo-sensitizing effect; Chemotherapy resistance; Metformin; Mitochondrial transfer
  11. Ann Hematol. 2022 Feb 07.
      The frequency of thrombosis in AML has been evaluated only in a few studies and no validated predictive model is currently available. Recently, DIC score was shown to identify patients at higher thrombotic risk. We aimed to evaluate the frequency of thromboembolism in AML patients treated with intensive chemotherapy and to assess the ability of genetic and clinical factors to predict the thrombotic risk. We performed a retrospective observational study including 222 newly diagnosed adult AML (210) and high-risk MDS (12), treated with intensive chemotherapy between January 2013 and February 2020. With a median follow-up of 44 months, we observed 50 thrombotic events (90% were venous, VTE). The prevalence of thrombosis was 22.1% and the 6-months cumulative incidence of thrombosis was 10%. The median time to thrombosis was 84 days and 52% of the events occurred within 100 days from AML diagnosis. Khorana and DIC score failed to stratify patients according to their thrombotic risk. Only history of a thrombotic event (p = 0.043), particularly VTE (p = 0.0053), platelet count above 100 × 109/L at diagnosis (p = 0.036) and active smoking (p = 0.025) significantly and independently increased the risk of thrombosis, the latter particularly of arterial events. AML genetic profile did not affect thrombosis occurrence. Results were confirmed considering only thromboses occurring within day 100 from diagnosis. DIC score at diagnosis, but not thrombosis, was independently associated with reduced survival (p = 0.004). Previous VTE, platelet count above 100 × 109/L and active smoking were the only factors associate with increased thrombotic risk in AML patients treated intensively, but further studies are needed to validate these results.
    Keywords:  Acute myeloid leukemia; Disseminated intravascular coagulation; Intensive chemotherapy; Khorana score; Thrombosis
  12. Am J Hematol. 2022 Feb 12.
      BACKGROUND: Post therapy measurable residual disease (MRD) positivity in core binding factor (CBF) AML is associated with shorter relapse free survival (RFS). Elimination of MRD measured via quantitative reverse transcription polymerase chain reaction (qRTPCR) for disease specific transcripts can potentially lead to better outcomes in CBF AML.METHODS: We prospectively monitored the MRD using qRTPCR and flow cytometry on bone marrow samples in patients with newly diagnosed CBF-AML who received decitabine (DAC) maintenance therapy after fludarabine/cytarabine/G-CSF (FLAG) based induction/consolidation regimen. Negative qRTPCR (CMR) was defined as fusion transcript <0.01% RESULTS: Thirty-one patients with CBF-AML including 14 with t (8; 21) and 17 with inv(16) received parenteral decitabine as maintenance therapy. Fifteen patients (48.3%) had completed FLAG based induction/consolidation but with positive MRD (0.35%, range 0.01-0.91%) (group 1). Sixteen patients (51.7%) could not complete recommended consolidations with FLAG based regimen (due to older age or complications) and were switched to decitabine maintenance (group 2). In group 2, 8 patients (50%) had undetectable MRD (group 2A) (all had qRTPCR ≤0.01%) and the other 8 patients (50%) had residual fusion product by qRTPCR (0.1%, range 0.02-0.36%) (group 2B) prior to starting decitabine Amongst the 23 patients who had a PCR ≥0.01% before maintenance therapy (Group1 and 2B), 12 patients (52%), (67% patients in Group 1 and 25% patients in Group 2B) attained a CMR (qRTPCR <0.01%) as their best response (responders). The median pre-DAC qRTPCR amongst responders were 0.03% compared to 0.14% in non-responders (P = 0.002). Overall, 20 patients attained CMR with DAC, of whom 15 remained in CMR after the completion of decitabine maintenance. The median estimated molecular relapse free survival amongst responders were 93.9 months. Only 5/31(16%) patients had a hematological relapse while on DAC maintenance, 1 each in Groups 1 and 2A and 3 in Group 2B. At a median follow up of 59.3 months (13.2-106 months) from DAC initiation, sixteen patients (51.6%) had to be initiated on a second line of therapy (40%, 25% and 100% patients respectively in Groups1, 2A and 2B). The median estimated time to new treatment between responders was 112.4 months vs. 5.8 months in non-responders (HR = 0.16, 95% CI = 0.04-0.54); however, there were no difference in OS between these groups (P = 0.37) CONCLUSION: Decitabine is an effective maintenance therapy for CBF-AML patients with persistent fusion transcript at a low level after FLAG based regimen. Attainment of CMR with DAC maintenance can lead to long term remission in patients who have persistent MRD positive after FLAG based regimen or are unable to receive the full course of consolidation therapy This article is protected by copyright. All rights reserved.
  13. Sci Adv. 2022 Feb 11. 8(6): eabg9455
      Super Enhancers (SEs) are clusters of regulatory elements associated with cell identity and disease. However, whether these elements are induced by oncogenes and can regulate gene modules cooperating for cancer cell transformation or maintenance remains elusive. To address this question, we conducted a genome-wide CRISPRi-based screening of SEs in ETO2-GLIS2+ acute megakaryoblastic leukemia. This approach revealed SEs essential for leukemic cell growth and survival that are induced by ETO2-GLIS2 expression. In particular, we identified a de novo SE specific of this leukemia subtype and regulating expression of tyrosine kinase-associated receptors KIT and PDGFRA. Combined expression of these two receptors was required for leukemic cell growth, and CRISPRi-mediated inhibition of this SE or treatment with tyrosine kinase inhibitors impaired progression of leukemia in vivo in patient-derived xenografts experiments. Our results show that fusion oncogenes, such as ETO2-GLIS2, can induce activation of SEs regulating essential gene modules synergizing for leukemia progression.
  14. Blood. 2022 Feb 08. pii: blood.2021015161. [Epub ahead of print]
      The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We employed a novel approach to prioritize immunotherapy targets and this identified a cell surface protein not previously implicated in myeloma, SEMA4A. Using knock-down by shRNA and CRISPR/dCas9, we demonstrate that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.
  15. Sci Transl Med. 2022 Feb 09. 14(631): eabg8070
      Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.
  16. Cancer Sci. 2022 Feb 08.
      Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T cell infiltration at tumor sites and programmed death receptor-1(PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T cell dysregulation, including aberrant intrathymic T cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T cell development and function, which contributes to creating a pro-tumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid tumor progression in ASXL1-CHIP carriers.
    Keywords:  ASXL1; CHIP; Mouse solid tumor models; T-cell; Tumor immunity
  17. Sci Transl Med. 2022 Feb 09. 14(631): eabg8027
      T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.
  18. Blood Adv. 2022 Feb 10. pii: bloodadvances.2021006357. [Epub ahead of print]
      DNA methyltransferase inhibitors (DNMTIs) for patients with higher risk myelodysplastic syndromes (HR-MDS) have low complete remission rates and are not curative. Early DNMTI combination clinical trials in HR-MDS are often termed "promising", but many randomized trials subsequently failed to show benefit. Clearer understanding when a combination is likely to improve upon DNMTI monotherapy would inform randomized studies. We reviewed MDS azacitidine or decitabine monotherapy studies. We collected baseline demographics including IPSS risk, DNMTI, disease characteristics; and response variables including survival, marrow and hematologic responses. Aggregate estimates across studies were calculated using meta-analyses techniques. Using a binomial design, we estimated the necessary operating characteristics to design a phase II study showing improved efficacy of a combination over monotherapy. Among 1908 patients the overall response rate ORR was 24% (n=464, 95%CI 0.22-0.26): 267 complete response (CR, 14%), 68 partial response (PR, 4%) and 129 marrow complete remission (mCR, 7%). Among 1604 patients for whom a hematologic response was reported, 476 patients (30%, 95%CI 0.27-0.32) reported hematologic improvement (HI). More patients treated with azacitidine achieved HI (38%, 95%CI 0.35-0.41) compared to decitabine (15%, 95%CI 0.13-0.19), while the marrow ORR rate was higher with decitabine (29%,95%CI 0.26-0.33) compared to azacitidine (21%, 95%CI 0.19-0.23). CR rates were similar between DNMTIs: 13% with azacitidine and 16% with decitabine. Variables that influence MDS response include the specific DNMTI backbone and the distribution of IPSS risk of patients enrolled on a trial. Considering these factors can help identify which early combination approaches are worth assessing in larger randomized trials.
  19. Nat Genet. 2022 Feb;54(2): 128-133
      The infinite sites model of molecular evolution posits that every position in the genome is mutated at most once1. By restricting the number of possible mutation histories, haplotypes and alleles, it forms a cornerstone of tumor phylogenetic analysis2 and is often implied when calling, phasing and interpreting variants3,4 or studying the mutational landscape as a whole5. Here we identify 18,295 biallelic mutations, where the same base is mutated independently on both parental copies, in 559 (21%) bulk sequencing samples from the Pan-Cancer Analysis of Whole Genomes study. Biallelic mutations reveal ultraviolet light damage hotspots at E26 transformation-specific (ETS) and nuclear factor of activated T cells (NFAT) binding sites, and hypermutable motifs in POLE-mutant and other cancers. We formulate recommendations for variant calling and provide frameworks to model and detect biallelic mutations. These results highlight the need for accurate models of mutation rates and tumor evolution, as well as their inference from sequencing data.
  20. Blood Rev. 2022 Feb 02. pii: S0268-960X(22)00006-6. [Epub ahead of print] 100932
      The role of the vascular microenvironment is increasingly studied in acute myeloid leukaemia (AML). Complex interactions between endothelial cells (ECs) and pre-leukaemic cells may contribute to the clonal evolution of pre-leukaemic stem cells in the bone marrow niche and to the proliferation, survival and chemoresistance of leukaemic cells. Through the expression of different adhesion molecules, ECs play a key role in the development of specific acute complications of AML, including leukostasis, acute respiratory failure, acute kidney injury or neurological complications. Moreover, in newly diagnosed patients, leukaemic cells promote endothelial activation and subsequent disseminated intravascular coagulation. Mechanisms of this bi-directional dialogue between leukaemic cells and ECs will reveal possible therapeutic targets to be explored to improve the survival of AML patients.
    Keywords:  Acute myeloid leukaemia; Endothelial cell; Hematopoietic stem cell
  21. Blood Adv. 2022 Feb 07. pii: bloodadvances.2021006889. [Epub ahead of print]
      Ruxolitinib (RUX) is extensively used in myelofibrosis (MF). Despite its early efficacy, most patients lose response over time and, after discontinuation, have a worse overall survival (OS). Currently, response criteria able to predict OS in RUX-treated patients are lacking, leading to uncertainty regarding the switch to second-line treatments. In this study, we investigated predictors of survival collected after six months of RUX in 209 MF patients participating in the real-world ambispective observational RUXOREL-MF study (NCT03959371). Multivariable analysis identified the following risk factors: (i) RUX dose <20 mg twice daily at baseline, month 3, and 6 (hazard ratio, HR, 1.79, 95% confidence interval, CI, 1.07-3.00, p=0.03), (ii) palpable spleen length reduction from baseline ≤30% at month 3 and 6 (HR 2.26, 95% CI 1.40-3.65, p=0.0009), (iii) red blood cell (RBC) transfusion need at month 3 and/or 6 (HR 1.66, 95% CI 0.95-2.88, p=0.07), and (iv) RBC transfusion need at all time points, i.e. baseline and months 3 and 6 (HR 2.32, 95% CI 1.19-4.54, p=0.02). Hence, we developed a prognostic model, named Response to Ruxolitinib after 6 months (RR6), dissecting three risk categories: low (median OS not reached), intermediate (median OS 61 months, 95% CI 43-80), and high (median OS 33 months, 95% CI 21-50). The RR6 model was validated and confirmed in an external cohort comprised of 40 MF patients. In conclusion, the RR6 prognostic model allows for the early identification of RUX-treated MF patients with impaired survival who might benefit from a prompt treatment shift.