bims-tremyl 2021-08-08 papers

bims-tremyl

Biomed News

on Therapy resistance biology in myeloid leukemia

Issue of 2021–08–08
twenty papers selected by
Paolo Gallipoli, Barts Cancer Institute, Queen Mary University of London

MICA/B antibody induces macrophage-mediated immunity against acute myeloid leukemia.
Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.
Targeting leukemia-specific dependence on the de novo purine synthesis pathway.
NOVEL NPM1 EXON 5 MUTATIONS AND GENE FUSIONS LEADING TO ABERRANT CYTOPLASMIC NUCLEOPHOSMIN IN AML.
Development of TP53 Mutations Over the Course of Therapy for Acute Myeloid Leukemia.
Ten-day decitabine with venetoclax versus intensive chemotherapy in relapsed or refractory acute myeloid leukemia: A propensity score-matched analysis.
Transcription Factors, R-Loops and Deubiquitinating Enzymes: Emerging Targets in Myelodysplastic Syndromes and Acute Myeloid Leukemia.
Multisite 11-year experience of less-intensive vs intensive therapies in acute myeloid leukemia.
High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.
ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency.
ANP32B-mediated repression of p53 contributes to maintenance of normal and CML stem cells.
TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.
Phase II study of azacitidine with pembrolizumab in patients with intermediate-1 or higher-risk myelodysplastic syndrome.
Bleeding diathesis in mice lacking JAK2 in platelets.
Clonal expansion of T memory stem cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients.
Clonal hematopoiesis in patients receiving chimeric antigen receptor T-cell therapy.
Trametinib enhances ATRA-induced differentiation in AML cells.
The role of reciprocal fusions in MLL-r acute leukemia: studying the chromosomal translocation t(6;11).
Clinical outcome of chronic myeloid leukemia patients who switch from first-line therapy with a second generation tyrosine kinase inhibitor to an alternative TKI.
METTL1-mediated m7G modification of Arg-TCT tRNA drives oncogenic transformation.

Blood. 2021 Aug 06. pii: blood.2021011619. [Epub ahead of print]

MICA/B antibody induces macrophage-mediated immunity against acute myeloid leukemia.

Pedro Henrique Alves da Silva, Samantha Xing, Andriana G Kotini, Eirini P Papapetrou, Xiaoyu Song, Kai W Wucherpfennig, John Mascarenhas, Lucas Ferrari de Andrade.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem and progenitor cell malignancy characterized by poor clinical outcomes. MICA and MICB (MICA/B) are stress-proteins expressed by cancer cells, and antibody-mediated inhibition of MICA/B shedding represents a novel approach to stimulate immunity against cancers. We found that the MICA/B antibody 7C6 potently inhibits the outgrowth of AML in two models in immunocompetent mice. Macrophages were essential for therapeutic efficacy, and 7C6 triggered antibody-dependent phagocytosis of AML cells. Furthermore, we found that romidepsin, a selective histone deacetylase inhibitor, increased MICB mRNA in AML cells and enabled subsequent stabilization of the translated protein by 7C6. This drug combination substantially increased surface MICA/B expression in a human AML line, pluripotent stem cell-derived AML blasts and leukemia stem cells, as well as primary cells from three untreated AML patients. Human macrophages phagocytosed AML cells following treatment with 7C6 and romidepsin, and the combination therapy lowered leukemia burden in a humanized model of AML. Therefore, inhibition of MICA/B shedding promotes macrophage-driven immunity against AML via Fc receptor signaling and synergizes with an epigenetic regulator. These results provide the rationale for the clinical testing of this innovative immunotherapeutic approach for the treatment of AML.

DOI: https://doi.org/10.1182/blood.2021011619
Blood. 2021 Aug 25. pii: blood.2021012629. [Epub ahead of print]

Targeting RIOK2 ATPase activity leads to decreased protein synthesis and cell death in acute myeloid leukemia.

Jan-Erik Messling, Karl Agger, Kasper L Andersen, Kristina Kromer, Hanna Maria Kuepper, Anders H Lund, Kristian Helin.

Novel therapies for the treatment of acute myeloid leukemia (AML) are urgently needed as current treatments do not cure the majority of AML patients. Here, we report on a domain-focused, kinome-wide CRISPR-Cas9 screen to identify protein kinase targets for the treatment of AML, which led to the identification of Rio-kinase 2 (RIOK2) as a potential novel target. We show that loss of RIOK2 leads to a decrease in protein synthesis and to ribosomal instability followed by apoptosis in leukemic cells, but not in fibroblasts. Moreover, we demonstrate that the ATPase function of RIOK2 is required for cell survival. By using a small molecule inhibitor, we show that pharmacological inhibition of RIOK2 similarly leads to loss of protein synthesis and apoptosis and affects leukemic cell growth in vivo. Our results provide proof-of-concept for targeting RIOK2 as a potential treatment for AML patients.

DOI: https://doi.org/10.1182/blood.2021012629
Leukemia. 2021 Aug 03.

Targeting leukemia-specific dependence on the de novo purine synthesis pathway.

Takuji Yamauchi, Kohta Miyawaki, Yuichiro Semba, Masatomo Takahashi, Yoshihiro Izumi, Jumpei Nogami, Fumihiko Nakao, Takeshi Sugio, Kensuke Sasaki, Luca Pinello, Daniel E Bauer, Takeshi Bamba, Koichi Akashi, Takahiro Maeda.

Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival.

DOI: https://doi.org/10.1038/s41375-021-01369-0
Blood. 2021 08 03. pii: blood.2021012732. [Epub ahead of print]

NOVEL NPM1 EXON 5 MUTATIONS AND GENE FUSIONS LEADING TO ABERRANT CYTOPLASMIC NUCLEOPHOSMIN IN AML.

Maria Paola Martelli, Roberta Rossi, Alessandra Venanzi, Manja Meggendorfer, Vincenzo Maria Perriello, Giovanni Martino, Orietta Spinelli, Raffaella Ciurnelli, Emanuela Varasano, Lorenzo Brunetti, Stefano Ascani, Corinne Quadalti, Valeria Cardinali, Federica Mezzasoma, Ilaria Gionfriddo, Francesca Milano, Roberta Pacini, Alessia Tabarrini, Barbara Bigerna, Francesco Albano, Giorgina Specchia, Calogero Vetro, Francesco Di Raimondo, Ombretta Annibali, Giuseppe Avvisati, Alessandro Rambaldi, Franca Falzetti, Enrico Tiacci, Paolo Sportoletti, Torsten Haferlach, Claudia Haferlach, Brunangelo Falini.

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but sporadically also exon 9 and 11, all causing changes at protein C-terminal end (loss of tryptophans and creation of a nuclear export signal-NES motif) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 AML patients, we found non-exon 12 NPM1 mutations in 5/387 (1.3%) NPM1c+ cases. Besides mutations in exon 9 (n=1) and exon 11 (n=1), novel mutations in exon 5 were discovered (n=3). One more exon 5 mutation was identified in additional 141 AML patients selected for wild-type NPM1 exon 12. Furthermore, 3 NPM1 rearrangements (i.e. NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13,979 AML samples screened by cytogenetic/FISH and RNA sequencing. Functional studies demonstrated that in AML cases the new NPM1 proteins harboured an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting any AML-associated NPM1 genetic lesions. Also, this study highlights the need for developing new specific assays for molecular diagnosis and monitoring of NPM1-mutated AML.

DOI: https://doi.org/10.1182/blood.2021012732
Am J Hematol. 2021 Aug 05.

Development of TP53 Mutations Over the Course of Therapy for Acute Myeloid Leukemia.

Yasmin Alwash, Joseph D Khoury, Mehrnoosh Tashakori, Rashmi Kanagal-Shamanna, Naval Daver, Farhad Ravandi, Tapan M Kadia, Marina Konopleva, Courtney D Dinardo, Ghayas C Issa, Sanam Loghavi, Koichi Takahashi, Elias Jabbour, Veronica Guerra, Steven Kornblau, Hagop Kantarjian, Nicholas J Short.

TP53 mutations in acute myeloid leukemia (AML) are associated with resistance to standard treatments and dismal outcomes. The incidence and prognostic impact of the emergence of newly detectable TP53 mutations over the course of AML therapy has not been well described. We retrospectively analyzed 200 patients with newly diagnosed TP53 wild type AML who relapsed after or were refractory to frontline therapy. Twenty-nine patients (15%) developed a newly detectable TP53 mutation in the context of relapsed/refractory disease. The median variant allelic frequency (VAF) was 15% (range, 1.1% - 95.6%). TP53 mutations were more common after intensive therapy versus lower-intensity therapy (23% versus 10%, respectively; P=0.02) and in patients who had undergone hematopoietic stem cell transplant versus those who had not (36% versus 12%, respectively; P=0.005). Lower TP53 VAF was associated with an increased likelihood of complete remission (CR) or CR with incomplete hematologic recovery (CRi) compared to higher TP53 VAF (CR/CRi rate of 41% for VAF <20% versus 13% for VAF ≥ 20%, respectively). The median overall survival (OS) after acquisition of TP53 mutation was 4.6 months, with a 1-year OS rate of 19%. TP53 VAF at relapse was significantly associated with OS; the median OS of patients with TP53 VAF ≥20% was 3.5 months versus 6.1 months for those with TP53 VAF <20% (P<0.05). In summary, new TP53 mutations may be acquired throughout the course of AML therapy. Sequential monitoring for TP53 mutations is likely to be increasingly relevant in the era of emerging TP53-targeting therapies for AML. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1002/ajh.26314
Cancer. 2021 Aug 03.

Ten-day decitabine with venetoclax versus intensive chemotherapy in relapsed or refractory acute myeloid leukemia: A propensity score-matched analysis.

Abhishek Maiti, Courtney D DiNardo, Wei Qiao, Tapan M Kadia, Elias J Jabbour, Caitlin R Rausch, Naval G Daver, Nicholas J Short, Gautam Borthakur, Naveen Pemmaraju, Musa Yilmaz, Yesid Alvarado, Kathryn S Montalbano, Allison Wade, Rita E Maduike, Julio A Guerrero, Kenneth Vaughan, Carol A Bivins, Sherry Pierce, Jing Ning, Farhad Ravandi, Hagop M Kantarjian, Marina Y Konopleva.

   BACKGROUND: Relapsed/refractory (R/R) acute myeloid leukemia (AML) has poor outcomes. Although lower-intensity venetoclax-containing regimens are standard for older/unfit patients with newly diagnosed AML, it is unknown how such regimens compare with intensive chemotherapy (IC) for R/R AML.
METHODS: Outcomes of R/R AML treated with 10-day decitabine and venetoclax (DEC10-VEN) were compared with IC-based regimens including idarubicin with cytarabine, with or without cladribine, clofarabine, or fludarabine, with or without additional agents. Propensity scores derived from patient baseline characteristics were used to match DEC10-VEN and IC patients to minimize bias.
RESULTS: Sixty-five patients in the DEC10-VEN cohort were matched to 130 IC recipients. The median ages for the DEC10-VEN and IC groups were 64 and 58 years, respectively, and baseline characteristics were balanced between the 2 cohorts. DEC10-VEN conferred significantly higher responses compared with IC including higher overall response rate (60% vs 36%; odds ratio [OR], 3.28; P < .001), complete remission with incomplete hematologic recovery (CRi, 19% vs 6%; OR, 3.56; P = .012), minimal residual disease negativity by flow cytometry (28% vs 13%; OR, 2.48; P = .017), and lower rates of refractory disease. DEC10-VEN led to significantly longer median event-free survival compared with IC (5.7 vs 1.5 months; hazard ratio [HR], 0.46; 95% CI, 0.30-0.70; P < .001), as well as median overall survival (OS; 6.8 vs 4.7 months; HR, 0.56; 95% CI, 0.37-0.86; P = .008). DEC10-VEN was independently associated with improved OS compared with IC in multivariate analysis. Exploratory analysis for OS in 27 subgroups showed that DEC10-VEN was comparable with IC as salvage therapy for R/R AML.
CONCLUSION: DEC10-VEN represents an appropriate salvage therapy and may offer better responses and survival compared with IC in adults with R/R AML.

Keywords:  acute myeloid leukemia (AML); decitabine; intensive chemotherapy; refractory; relapsed; venetoclax

DOI:  https://doi.org/10.1002/cncr.33814
Cancers (Basel). 2021 Jul 26. pii: 3753. [Epub ahead of print]13(15):

Transcription Factors, R-Loops and Deubiquitinating Enzymes: Emerging Targets in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Silvia M L Barabino, Elisabetta Citterio, Antonella Ellena Ronchi.

  Myeloid neoplasms encompass a very heterogeneous family of diseases characterized by the failure of the molecular mechanisms that ensure a balanced equilibrium between hematopoietic stem cells (HSCs) self-renewal and the proper production of differentiated cells. The origin of the driver mutations leading to preleukemia can be traced back to HSC/progenitor cells. Many properties typical to normal HSCs are exploited by leukemic stem cells (LSCs) to their advantage, leading to the emergence of a clonal population that can eventually progress to leukemia with variable latency and evolution. In fact, different subclones might in turn develop from the original malignant clone through accumulation of additional mutations, increasing their competitive fitness. This process ultimately leads to a complex cancer architecture where a mosaic of cellular clones-each carrying a unique set of mutations-coexists. The repertoire of genes whose mutations contribute to the progression toward leukemogenesis is broad. It encompasses genes involved in different cellular processes, including transcriptional regulation, epigenetics (DNA and histones modifications), DNA damage signaling and repair, chromosome segregation and replication (cohesin complex), RNA splicing, and signal transduction. Among these many players, transcription factors, RNA splicing proteins, and deubiquitinating enzymes are emerging as potential targets for therapeutic intervention.

Keywords:  R-loops; RNA splicing; acute myeloid leukemia (AML); deubiquitinating enzymes (DUBs); genome integrity; myelodysplastic syndromes (MDS); transcription factors

DOI:  https://doi.org/10.3390/cancers13153753
Blood. 2021 Aug 05. 138(5): 387-400

Multisite 11-year experience of less-intensive vs intensive therapies in acute myeloid leukemia.

Mohamed L Sorror, Barry E Storer, Amir T Fathi, Andrew Brunner, Aaron T Gerds, Mikkael A Sekeres, Sudipto Mukherjee, Bruno C Medeiros, Eunice S Wang, Pankit Vachhani, Paul J Shami, Esteban Peña, Mahmoud Elsawy, Kehinde Adekola, Selina Luger, Maria R Baer, David Rizzieri, Tanya M Wildes, Jamie Koprivnikar, Julie Smith, Mitchell Garrison, Kiarash Kojouri, Wendy Leisenring, Lynn Onstad, Jennifer E Nyland, Pamela S Becker, Jeannine S McCune, Stephanie J Lee, Brenda M Sandmaier, Frederick R Appelbaum, Elihu H Estey.

Less-intensive induction therapies are increasingly used in older patients with acute myeloid leukemia (AML). Using an AML composite model (AML-CM) assigning higher scores to older age, increased comorbidity burdens, and adverse cytogenetic risks, we defined 3 distinct prognostic groups and compared outcomes after less-intensive vs intensive induction therapies in a multicenter retrospective cohort (n = 1292) treated at 6 institutions from 2008 to 2012 and a prospective cohort (n = 695) treated at 13 institutions from 2013 to 2017. Prospective study included impacts of Karnofsky performance status (KPS), quality of life (QOL), and physician perception of cure. In the retrospective cohort, recipients of less-intensive therapies were older and had more comorbidities, more adverse cytogenetics, and worse KPS. Less-intensive therapies were associated with higher risks of mortality in AML-CM scores of 4 to 6, 7 to 9, and ≥10. Results were independent of allogeneic transplantation and similar in those age 70 to 79 years. In the prospective cohort, the 2 groups were similar in baseline QOL, geriatric assessment, and patient outcome preferences. Higher mortality risks were seen after less-intensive therapies. However, in models adjusted for age, physician-assigned KPS, and chance of cure, mortality risks and QOL were similar. Less-intensive therapy recipients had shorter length of hospitalization (LOH). Our study questions the survival and QOL benefits (except LOH) of less-intensive therapies in patients with AML, including those age 70 to 79 years or with high comorbidity burdens. A randomized trial in older/medically infirm patients is required to better assess the value of less-intensive and intensive therapies or their combination. This trial was registered at www.clinicaltrials.gov as #NCT01929408.

DOI: https://doi.org/10.1182/blood.2020008812
Blood Cancer J. 2021 Jul 31. 11(7): 137

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.

Ruochen Jia, Leon Kutzner, Anna Koren, Kathrin Runggatscher, Peter Májek, André C Müller, Michael Schuster, Christoph Bock, Joanna I Loizou, Stefan Kubicek, Robert Kralovics.

Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

DOI: https://doi.org/10.1038/s41408-021-00531-2
Mol Cell. 2021 Jul 28. pii: S1097-2765(21)00587-6. [Epub ahead of print]

ZMYND8-regulated IRF8 transcription axis is an acute myeloid leukemia dependency.

Zhendong Cao, Krista A Budinich, Hua Huang, Diqiu Ren, Bin Lu, Zhen Zhang, Qingzhou Chen, Yeqiao Zhou, Yu-Han Huang, Fatemeh Alikarami, Molly C Kingsley, Alexandra K Lenard, Aoi Wakabayashi, Eugene Khandros, Will Bailis, Jun Qi, Martin P Carroll, Gerd A Blobel, Robert B Faryabi, Kathrin M Bernt, Shelley L Berger, Junwei Shi.

  The transformed state in acute leukemia requires gene regulatory programs involving transcription factors and chromatin modulators. Here, we uncover an IRF8-MEF2D transcriptional circuit as an acute myeloid leukemia (AML)-biased dependency. We discover and characterize the mechanism by which the chromatin "reader" ZMYND8 directly activates IRF8 in parallel with the MYC proto-oncogene through their lineage-specific enhancers. ZMYND8 is essential for AML proliferation in vitro and in vivo and associates with MYC and IRF8 enhancer elements that we define in cell lines and in patient samples. ZMYND8 occupancy at IRF8 and MYC enhancers requires BRD4, a transcription coactivator also necessary for AML proliferation. We show that ZMYND8 binds to the ET domain of BRD4 via its chromatin reader cassette, which in turn is required for proper chromatin occupancy and maintenance of leukemic growth in vivo. Our results rationalize ZMYND8 as a potential therapeutic target for modulating essential transcriptional programs in AML.

Keywords:  IRF8; MEF2D; ZMYND8; acute myeloid leukemia; epigenetics; transcriptional addiction

DOI:  https://doi.org/10.1016/j.molcel.2021.07.018
Blood. 2021 Aug 06. pii: blood.2020010400. [Epub ahead of print]

ANP32B-mediated repression of p53 contributes to maintenance of normal and CML stem cells.

Shuo Yang, Xiao-Na Zhu, Hui-Lin Zhang, Qian Yang, Yu-Sheng Wei, Di Zhu, Meng-Di Liu, Shao-Ming Shen, Li Xia, Ping He, Meng-Kai Ge, Yi-Lian Pan, Meng Zhao, Yingli Wu, Junke Zheng, Guo-Qiang Chen, Yun Yu.

Proper regulation of p53 signaling is critical for the maintenance of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). The hematopoietic cell-specific mechanisms regulating p53 activity remain largely unknown. Here, we demonstrate that conditional deletion of acidic leucine-rich nuclear phosphoprotein 32B (ANP32B) in hematopoietic cells impairs repopulation capacity and post-injury regeneration of HSCs. Mechanistically, ANP32B forms a repressive complex with and thus inhibits the transcriptional activity of p53 in hematopoietic cells, and p53 deletion rescues the functional defect in Anp32b-deficient HSCs. Of great interest, ANP32B is highly expressed in leukemic cells from chronic myelogenous leukemia (CML) patients. Anp32b deletion enhances p53 transcriptional activity to impair LSCs function in a murine CML model, and exhibits synergistic therapeutic effects with tyrosine kinase inhibitors in inhibiting CML propagation. In summary, our findings provide a novel strategy to enhance p53 activity in LSCs by inhibiting ANP32B, and identify ANP32B as a potential therapeutic target in treating CML.

DOI: https://doi.org/10.1182/blood.2020010400
Cell Stem Cell. 2021 Jul 28. pii: S1934-5909(21)00288-5. [Epub ahead of print]

TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.

Laura García-Prat, Kerstin B Kaufmann, Florin Schneiter, Veronique Voisin, Alex Murison, Jocelyn Chen, Michelle Chan-Seng-Yue, Olga I Gan, Jessica L McLeod, Sabrina A Smith, Michelle C Shoong, Darrien Parris, Kristele Pan, Andy G X Zeng, Gabriela Krivdova, Kinam Gupta, Shin-Ichiro Takayanagi, Elvin Wagenblast, Weijia Wang, Mathieu Lupien, Timm Schroeder, Stephanie Z Xie, John E Dick.

  It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.

Keywords:  MYC; TFEB; TfR1; anabolism; endocytosis; erythropoiesis; long-term HSC; lysosomes; myelopoiesis; self-renewal

DOI:  https://doi.org/10.1016/j.stem.2021.07.003
Br J Haematol. 2021 Aug 02.

Phase II study of azacitidine with pembrolizumab in patients with intermediate-1 or higher-risk myelodysplastic syndrome.

Kelly S Chien, Kunhwa Kim, Graciela M Nogueras-Gonzalez, Gautam Borthakur, Kiran Naqvi, Naval G Daver, Guillermo Montalban-Bravo, Jorge E Cortes, Courtney D DiNardo, Elias Jabbour, Yesid Alvarado, Michael Andreeff, Prithviraj Bose, Nitin Jain, Tapan M Kadia, Xuelin Huang, Kimberly B Sheppard, Cheri Klingner-Winton, Sherry A Pierce, Xiao Qin Dong, Kelly A Soltysiak, Hagop M Kantarjian, Guillermo Garcia-Manero.

  Programmed cell death protein 1 (PD-1) and PD-ligand 1 (PD-L1) expression is upregulated in cluster of differentiation 34 (CD34)+ bone marrow cells from patients with myelodysplastic syndromes (MDS). Hypomethylating agent (HMA) treatment results in further increased expression of these immune checkpoints. We hypothesised that combining an anti-PD-1 antibody with HMAs may have efficacy in patients with MDS. To test this concept, we designed a phase II trial of the combination of azacitidine and pembrolizumab with two cohorts. In the 17 previously untreated patients, the overall response rate (ORR) was 76%, with a complete response (CR) rate of 18% and median overall survival (mOS) not reached after a median follow-up of 12·8 months. For the HMA-failure cohort (n = 20), the ORR was 25% and CR rate was 5%; with a median follow-up of 6·0 months, the mOS was 5·8 months. The most observed toxicities were pneumonia (32%), arthralgias (24%) and constipation (24%). Immune-related adverse events requiring corticosteroids were required in 43%. Overall, this phase II trial suggests that azacitidine and pembrolizumab is safe with manageable toxicities in patients with higher-risk MDS. This combined therapy may have anti-tumour activity in a subset of patients and merits further studies in the front-line setting.

Keywords:  azacitidine; hypomethylating agent failure; immunotherapy; myelodysplastic syndromes; pembrolizumab

DOI:  https://doi.org/10.1111/bjh.17689
Blood Adv. 2021 Aug 10. 5(15): 2969-2981

Bleeding diathesis in mice lacking JAK2 in platelets.

Nathan Eaton, Saravanan Subramaniam, Marie L Schulte, Caleb Drew, David Jakab, Sandra L Haberichter, Hartmut Weiler, Hervé Falet.

The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells, such as hematopoietic stem cells and megakaryocytes (MKs). Individuals expressing the somatic JAK2 V617F mutation commonly develop myeloproliferative neoplasms (MPNs) associated with venous and arterial thrombosis, a leading cause of mortality. The role of JAK2 in hemostasis remains unclear. We investigated the role of JAK2 in platelet hemostatic function using Jak2fl/fl Pf4-Cre (Jak2Plt-/-) mice lacking JAK2 in platelets and MKs. Jak2Plt-/- mice developed MK hyperplasia and splenomegaly associated with severe thrombocytosis and bleeding. This notion was supported by failure to occlude in a ferric chloride carotid artery injury model and by a cremaster muscle laser-induced injury assay, in which Jak2Plt-/- platelets failed to form stable thrombi. Jak2Plt-/- platelets formed thrombi poorly after adhesion to type 1 collagen under arterial shear rates. Jak2Plt-/- platelets spread poorly on collagen under static conditions or on fibrinogen in response to the collagen receptor GPVI-specific agonist, collagen-related peptide (CRP). After activation with collagen, CRP, or the CLEC-2 agonist rhodocytin, Jak2Plt-/- platelets displayed decreased α-granule secretion and integrin αIIbβ3 activation or aggregation, but showed normal responses to thrombin. Jak2Plt-/- platelets had impaired intracellular signaling when activated via GPVI, as assessed by tyrosine phosphorylation. Together, the results show that JAK2 deletion impairs platelet immunoreceptor tyrosine-based activation motif signaling and hemostatic function in mice and suggest that aberrant JAK2 signaling in patients with MPNs affects GPVI signaling, leading to hemostatic platelet function.

DOI: https://doi.org/10.1182/bloodadvances.2020003032
Nat Cancer. 2021 Jun;2(6): 629-642

Clonal expansion of T memory stem cells determines early anti-leukemic responses and long-term CAR T cell persistence in patients.

Luca Biasco, Natalia Izotova, Christine Rivat, Sara Ghorashian, Rachel Richardson, Aleks Guvenel, Rachael Hough, Robert Wynn, Bilyana Popova, Andre Lopes, Martin Pule, Adrian J Thrasher, Persis J Amrolia.

Low-affinity CD19 chimeric antigen receptor (CAR) T cells display enhanced expansion and persistence, enabling fate tracking through integration site analysis. Here we show that integration sites from early (1 month) and late (>3yr) timepoints cluster separately, suggesting different clonal contribution to early responses and prolonged anti-leukemic surveillance. CAR T central and effector memory cells in patients with long-term persistence remained highly polyclonal, whereas diversity dropped rapidly in patients with limited CAR T persistence. Analysis of shared integrants between the CAR T cell product and post-infusion demonstrated that, despite their low frequency, T memory stem cell clones in the product contributed substantially to the circulating CAR T cell pools, during both early expansion and long-term persistence. Our data may help identify patients at risk of early loss of CAR T cells and highlight the critical role of T memory stem cells both in mediating early anti-leukemic responses and in long-term surveillance by CAR T cells.

DOI: https://doi.org/10.1038/s43018-021-00207-7
Blood Adv. 2021 Aug 10. 5(15): 2982-2986

Clonal hematopoiesis in patients receiving chimeric antigen receptor T-cell therapy.

Peter G Miller, Adam S Sperling, Elliott J Brea, Mark B Leick, Geoffrey G Fell, Max Jan, Satyen H Gohil, Yu-Tzu Tai, Nikhil C Munshi, Catherine J Wu, Donna S Neuberg, Marcela V Maus, Caron Jacobson, Christopher J Gibson, Benjamin L Ebert.

Chimeric antigen receptor (CAR) T-cells have emerged as an efficacious modality in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP), a state in which mutations in hematopoietic cells give rise to a clonal population of cells, is more common in patients exposed to cytotoxic therapies, has been shown to influence inflammatory immune programs, and is associated with an adverse prognosis in patients with NHL and MM receiving autologous transplantation. We therefore hypothesized that CHIP could influence clinical outcomes in patients receiving CAR T-cell therapy. In a cohort of 154 patients with NHL or MM receiving CAR T-cells, we found that CHIP was present in 48% of patients and associated with increased rates of complete response and cytokine release syndrome severity, but only in patients younger than age 60 years. Despite these differences, CHIP was not associated with a difference in progression-free or overall survival, regardless of age. Our data suggest that CHIP can influence CAR T-cell biology and clinical outcomes, but, in contrast to autologous transplantation, CHIP was not associated with worse survival and should not be a reason to exclude individuals from receiving this potentially life-prolonging treatment.

DOI: https://doi.org/10.1182/bloodadvances.2021004554
Leuk Lymphoma. 2021 Aug 06. 1-12

Trametinib enhances ATRA-induced differentiation in AML cells.

Hao Lu, Ze-Yi Li, Ming Ding, Cui Liang, Xiang-Qin Weng, Yan Sheng, Jing Wu, Xun Cai.

  All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib-ATRA (tra-ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra-ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.

Keywords:  Acute myeloid leukemia; all-trans retinoic acid; differentiation; trametinib

DOI:  https://doi.org/10.1080/10428194.2021.1961231
Oncogene. 2021 Aug 05.

The role of reciprocal fusions in MLL-r acute leukemia: studying the chromosomal translocation t(6;11).

Arpita Kundu, Eric Kowarz, Rolf Marschalek.

Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9-10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21-23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the "Massive Analyses of cDNA Ends" (MACE-Seq) technology, an established 3'-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.

DOI: https://doi.org/10.1038/s41388-021-01983-3
Leuk Res. 2021 Jul 24. pii: S0145-2126(21)00175-2. [Epub ahead of print]111 106674

Clinical outcome of chronic myeloid leukemia patients who switch from first-line therapy with a second generation tyrosine kinase inhibitor to an alternative TKI.

Chen-En Ma, Sunita Ghosh, Catherine Leyshon, Nikki Blosser, Deonne Dersch-Mills, Jennifer Jupp, Lynn Savoie, Elena Liew, Kareem Jamani.

  While second generation tyrosine kinase inhibitors (2GTKIs) are highly effective therapies for chronic myeloid leukemia (CML), a significant minority of patients who initiate a 2GTKI will require a switch to an alternative TKI. The long-term outcomes of those who require a change in therapy after front-line 2GTKI therapy are largely undescribed. Here we describe the clinical outcomes associated with switch to an alternative TKI after first-line therapy with a 2GTKI. Of 232 patients who initiated a 2GTKI during the study period, 76 (33 %) switched to an alternative TKI. Reasons for switching included intolerance (79 %) and resistance (21 %). Among the 60 patients who switched due to intolerance, 53 (88 %) were able to achieve or maintain a major molecular response (MMR) with 5-year progression-free survival (PFS) 90.5 % (95 % CI 90.4-90.6 %). Amongst the 16 patients who switched due to resistance, 8 patients (50 %) were able to achieve MMR with 5-year PFS 80.4 % (95 % CI 80.2-80.6 %). Most patients who switched due to intolerance remained on their second-line TKI. Approximately 25 % of patients who initiate first-line 2GTKI in a real world setting will ultimately switch to an alternate TKI due to intolerance. Patients who switch for intolerance continue to enjoy excellent clinical outcomes.

Keywords:  Chronic myeloid leukemia; Chronic phase chronic myeloid leukemia; Dasatinib; Nilotinib; Second generation tyrosine kinase inhibitor; Switch

DOI:  https://doi.org/10.1016/j.leukres.2021.106674
Mol Cell. 2021 Jul 29. pii: S1097-2765(21)00542-6. [Epub ahead of print]

METTL1-mediated m7G modification of Arg-TCT tRNA drives oncogenic transformation.

Esteban A Orellana, Qi Liu, Eliza Yankova, Mehdi Pirouz, Etienne De Braekeleer, Wencai Zhang, Jihoon Lim, Demetrios Aspris, Erdem Sendinc, Dimitrios A Garyfallos, Muxin Gu, Raja Ali, Alejandro Gutierrez, Sigitas Mikutis, Gonçalo J L Bernardes, Eric S Fischer, Allan Bradley, George S Vassiliou, Frank J Slack, Konstantinos Tzelepis, Richard I Gregory.

  The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.

Keywords:  Arg-TCT; METTL1; N(7)-methylguanosine; cancer; m(7)G; oncogene; tRNA; translation

DOI:  https://doi.org/10.1016/j.molcel.2021.06.031