bims-tremyl 2020-06-28 papers

on Therapy resistance biology in myeloid leukemia

Issue of 2020‒06‒28
twenty-one papers selected by
Paolo Gallipoli
Barts Cancer Institute, Queen Mary University of London

Male predominance in AML is associated with specific preleukemic mutations.
Synergistic Targeting of FLT3 Mutations in AML via Combined Menin-MLL and FLT3 Inhibition.
GSK3β suppression inhibits MCL1 protein synthesis in human acute myeloid leukemia cells.
Requirement for LIM kinases in acute myeloid leukemia.
Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor (CAR) T-cell Therapy for the Treatment of Acute Myeloid Leukemia.
The KDM Inhibitor GSKJ4 Triggers CREB Downregulation via a Protein Kinase A and Proteasome-Dependent Mechanism in Human Acute Myeloid Leukemia Cells.
Arsenic trioxide potentiates Gilteritinib-induced apoptosis in FLT3-ITD positive leukemic cells via IRE1a-JNK-mediated endoplasmic reticulum stress.
WT1 facilitates the self-renewal of leukemia-initiating cells through the upregulation of BCL2L2: WT1-BCL2L2 axis as a new acute myeloid leukemia therapy target.
Bosutinib for pretreated patients with chronic phase chronic myeloid leukemia: primary results of the phase 4 BYOND study.
Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.
Synergism between IL7R and CXCR4 drives BCR-ABL induced transformation in Philadelphia chromosome-positive acute lymphoblastic leukemia.
Targeting Bim via a lncRNA Morrbid Regulates the Survival of Preleukemic and Leukemic Cells.
Ex vivo drug screening defines novel drug sensitivity patterns for informing personalized therapy in myeloid neoplasms.
Lysine-specific demethylase 1A (LSD1) restricts ex vivo propagation of human HSCs and is a target of UM171.
FLT3 stop mutation increases FLT3 ligand level and risk of autoimmune thyroid disease.
The loss or absence of minimal residual disease of <0·1% at any time after two cycles of consolidation chemotherapy in CBFB-MYH11-positive acute myeloid leukaemia indicates poor prognosis.
Clonal dominance is an adverse prognostic factor in acute myeloid leukemia treated with intensive chemotherapy.
Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms.
PKCλ/ι Loss Induces Autophagy, Oxidative Phosphorylation, and NRF2 to Promote Liver Cancer Progression.
CARM1 inhibition reduces histone acetyltransferase activity causing synthetic lethality in CREBBP/EP300-mutated lymphomas.
Pervasive lesion segregation shapes cancer genome evolution.

Leukemia. 2020 Jun 23.

Male predominance in AML is associated with specific preleukemic mutations.

Aviv De-Morgan, Manja Meggendorfer, Claudia Haferlach, Liran Shlush.

DOI: https://doi.org/10.1038/s41375-020-0935-5
Blood. 2020 Jun 26. pii: blood.2020005037. [Epub ahead of print]

Synergistic Targeting of FLT3 Mutations in AML via Combined Menin-MLL and FLT3 Inhibition.

Margarita M Dzama, Marlene Steiner, Johanna Rausch, Daniel Sasca, Jonas Schönfeld, Kerstin Kunz, Martha C Taubert, Gerard M McGeehan, Chun-Wei Chen, Annalisa Mupo, Patricia S Hähnel, Matthias Theobald, Thomas Kindler, Richard Patrick Koche, George S Vassiliou, Scott A Armstrong, Michael W M Kühn.

The interaction of Menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and potential therapeutic opportunity against NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. Transcriptional profiling upon pharmacological inhibition of the Menin-MLL complex revealed specific changes in gene expression with downregulation of the MEIS1 transcription-factor and its transcriptional target gene FLT3 being most pronounced. Combining Menin-MLL-inhibition with specific small-molecule kinase inhibitors of FLT3-phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream to FLT3 signaling. The drug combination induced synergistic inhibition of proliferation as well as enhanced apoptosis and differentiation compared to single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary AML cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined Menin and FLT3-inhibition than to single-drug or vehicle control treatment, while AML cells with wildtype NPM1, MLL, and FLT3 were not affected by any of the two drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared to the single-drug and vehicle control groups. Our data suggest that combined Menin-MLL and FLT3-inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

DOI: https://doi.org/10.1182/blood.2020005037
J Cell Physiol. 2020 Jun 22.

GSK3β suppression inhibits MCL1 protein synthesis in human acute myeloid leukemia cells.

Yuan-Chin Lee, Yi-Jun Shi, Liang-Jun Wang, Jing-Ting Chiou, Chia-Hui Huang, Long-Sen Chang.

  Previous studies have shown that glycogen synthase kinase 3β (GSK3β) suppression is a potential strategy for human acute myeloid leukemia (AML) therapy. However, the cytotoxic mechanism associated with GSK3β suppression remains unresolved. Thus, the underlying mechanism of N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418)-elicited GSK3β suppression in the induction of AML U937 and HL-60 cell death was investigated in this study. Our study revealed that AR-A014418-induced MCL1 downregulation remarkably elicited apoptosis of U937 cells. Furthermore, the AR-A014418 treatment increased p38 MAPK phosphorylation and decreased the phosphorylated Akt and ERK levels. Activation of p38 MAPK subsequently evoked autophagic degradation of 4EBP1, while Akt inactivation suppressed mTOR-mediated 4EBP1 phosphorylation. Furthermore, AR-A014418-elicited ERK inactivation inhibited Mnk1-mediated eIF4E phosphorylation, which inhibited MCL1 mRNA translation in U937 cells. In contrast to GSK3α, GSK3β downregulation recapitulated the effect of AR-A014418 in U937 cells. Transfection of constitutively active GSK3β or cotransfection of constitutively activated MEK1 and Akt suppressed AR-A014418-induced MCL1 downregulation. Moreover, AR-A014418 sensitized U937 cells to ABT-263 (BCL2/BCL2L1 inhibitor) cytotoxicity owing to MCL1 suppression. Collectively, these results indicate that AR-A014418-induced GSK3β suppression inhibits ERK-Mnk1-eIF4E axis-modulated de novo MCL1 protein synthesis and thereby results in U937 cell apoptosis. Our findings also indicate a similar pathway underlying AR-A014418-induced death in human AML HL-60 cells.

Keywords:  4EBP1 downregulation; GSK3β suppression; MCL1 downregulation; eIF4E dephosphorylation; leukemia

DOI:  https://doi.org/10.1002/jcp.29884
Leukemia. 2020 Jun 26.

Requirement for LIM kinases in acute myeloid leukemia.

Patrizia Jensen, Michela Carlet, Richard F Schlenk, Andrea Weber, Jana Kress, Ines Brunner, Mikołaj Słabicki, Gregor Grill, Simon Weisemann, Ya-Yun Cheng, Irmela Jeremias, Claudia Scholl, Stefan Fröhling.

Acute myeloid leukemia (AML) is an aggressive disease for which only few targeted therapies are available. Using high-throughput RNA interference (RNAi) screening in AML cell lines, we identified LIM kinase 1 (LIMK1) as a potential novel target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients and coincided with FLT3 mutations, KMT2A rearrangements, and elevated HOX gene expression. RNAi- and CRISPR-Cas9-mediated suppression as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2 reduced colony formation and decreased proliferation due to slowed cell-cycle progression of KMT2A-rearranged AML cell lines and patient-derived xenograft (PDX) samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressor genes as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic basis for the anti-leukemic phenotype. Finally, we observed a reciprocal regulation between LIM kinases (LIMK) and CDK6, a kinase known to be involved in the differentiation block of KMT2A-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK inhibition. Together, these data suggest that LIMK are promising targets for AML therapy.

DOI: https://doi.org/10.1038/s41375-020-0943-5
Toxicol Sci. 2020 Jun 26. pii: kfaa098. [Epub ahead of print]

Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor (CAR) T-cell Therapy for the Treatment of Acute Myeloid Leukemia.

Christine Karbowski, Rebecca Goldstein, Brendon Frank, Kei Kim, Chi-Ming Li, Oliver Homann, Kelly Hensley, Benjamin Brooks, Xiaoting Wang, Qinghong Yan, Rocio Hernandez, Gregor Adams, Michael Boyle, Tara Arvedson, Herve Lebrec.

  FMS-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with two bispecific T cell engager (BiTE®) molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells while only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

Keywords:  3-6 chimeric antigen receptor T cells; acute myeloid leukemia; safety

DOI:  https://doi.org/10.1093/toxsci/kfaa098
Front Oncol. 2020 ;10 799

The KDM Inhibitor GSKJ4 Triggers CREB Downregulation via a Protein Kinase A and Proteasome-Dependent Mechanism in Human Acute Myeloid Leukemia Cells.

Michela Illiano, Mariarosaria Conte, Alessia Salzillo, Angela Ragone, Annamaria Spina, Angela Nebbioso, Lucia Altucci, Luigi Sapio, Silvio Naviglio.

  Acute myeloid leukemia (AML) is a progressive hematopoietic-derived cancer arising from stepwise genetic mutations of the myeloid lineage. cAMP response element-binding protein (CREB) is a nuclear transcription factor, which plays a key role in the multistep process of leukemogenesis, thus emerging as an attractive potential drug target for AML treatment. Since epigenetic dysregulations, such as DNA methylation, histone modifications, as well as chromatin remodeling, are a frequent occurrence in AML, an increasing and selective number of epi-drugs are emerging as encouraging therapeutic agents. Here, we demonstrate that the histone lysine demethylases (KDMs) JMJD3/UTX inhibitor GSKJ4 results in both proliferation decrease and CREB protein downregulation in AML cells. We found that GSKJ4 clearly decreases CREB protein, but not CREB mRNA levels. By cycloheximide assay, we provide evidence that GSKJ4 reduces CREB protein stability; moreover, proteasome inhibition largely counteracts the GSKJ4-induced CREB downregulation. Very interestingly, a rapid CREB phosphorylation at the Ser133 residue precedes CREB protein decrease in response to GSKJ4 treatment. In addition, protein kinase A (PKA) inhibition, but not extracellular signal-regulated kinase (ERK)1/2 inhibition, almost completely prevents both GSKJ4-induced p-Ser133-CREB phosphorylation and CREB protein downregulation. Overall, our study enforces the evidence regarding CREB as a potential druggable target, identifies the small epigenetic molecule GSKJ4 as an "inhibitor" of CREB, and encourages the design of future GSKJ4-based studies for the development of innovative approaches for AML therapy.

Keywords:  GSKJ4; acute myeloid leukemia (AML); cAMP response element-binding protein (CREB) downregulation; proteasome-mediated degradation; protein kinase A (PKA)

DOI:  https://doi.org/10.3389/fonc.2020.00799
Cancer Cell Int. 2020 ;20 250

Arsenic trioxide potentiates Gilteritinib-induced apoptosis in FLT3-ITD positive leukemic cells via IRE1a-JNK-mediated endoplasmic reticulum stress.

Xiaoli Hu, Jiayi Cai, Jianyi Zhu, Wenjing Lang, Jihua Zhong, Hua Zhong, Fangyuan Chen.

  Background: Acute myeloid leukemia (AML) patients with FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) have a high relapse rate and poor prognosis. This study aims to explore the underlying mechanism of combining Gilteritinib with ATO at low concentration in the treatment of FLT3-ITD positive leukemias.Methods: We used both in vitro and in vivo studies to investigate the effects of combination of Gilteritinib with ATO at low concentration on FLT3-ITD positive leukemias, together with the underlying molecular mechanisms of these processes.
Results: Combination of Gilteritinib with ATO showed synergistic effects on inhibiting proliferation, increasing apoptosis and attenuating invasive ability in FLT3-ITD-mutated cells and reducing tumor growth in nude mice. Results of western blot indicated that Gilteritinib increased a 160KD form of FLT3 protein on the surface of cell membrane. Detection of endoplasmic reticulum stress marker protein revealed that IRE1a and its downstream signal phosphorylated JNK were suppressed in Gilteritinib-treated FLT3-ITD positive cells. The downregulation of IRE1a induced by Gilteritinib was reversed with addition of ATO. Knockdown of IRE1a diminished the combinatorial effects of Gilteritinib plus ATO treatment and combination of tunicamycin (an endoplasmic reticulum pathway activator) with Gilteritinib achieved the similar effect as treatment with Gilteritinib plus ATO.
Conclusions: Thus, ATO at low concentration potentiates Gilteritinib-induced apoptosis in FLT3-ITD positive leukemic cells via IRE1a-JNK signal pathway, targeting IRE1a to cooperate with Gilteritinib may serve as a new theoretical basis on FLT3-ITD mutant AML treatment.

Keywords:  ATO; Endoplasmic reticulum stress; FLT3-ITD; Gilteritinib; IRE1a-JNK

DOI:  https://doi.org/10.1186/s12935-020-01341-5
J Transl Med. 2020 Jun 24. 18(1): 254

WT1 facilitates the self-renewal of leukemia-initiating cells through the upregulation of BCL2L2: WT1-BCL2L2 axis as a new acute myeloid leukemia therapy target.

Bin Zhou, Xianghong Jin, Weiwei Jin, Xingzhou Huang, Yanfei Wu, Haiying Li, Weijian Zhu, Xiaoyi Qin, Haige Ye, Shenmeng Gao.

  BACKGROUND: Overexpression of Wilms' tumor-1 (WT1) transcription factor facilitates proliferation in acute myeloid leukemia (AML). However, whether WT1 is enriched in the leukemia-initiating cells (LICs) and leukemia stem cells (LSCs) and facilitates the self-renewal of LSCs remains poorly understood.METHODS: MLL-AF9-induced murine leukemia model was used to evaluate the effect of knockdown of wt1 on the self-renewal ability of LSC. RNA sequencing was performed on WT1-overexpressing cells to select WT1 targets. Apoptosis and colony formation assays were used to assess the anti-leukemic potential of a deubiquitinase inhibitor WP1130. Furthermore, NOD/SCID-IL2Rγ (NSG) AML xenotransplantation and MLL-AF9-induced murine leukemia models were used to evaluate the anti-leukemogenic potential of WP1130 in vivo.
RESULTS: We found that wt1 is highly expressed in LICs and LSCs and facilitates the maintenance of leukemia in a murine MLL-AF9-induced model of AML. WT1 enhanced the self-renewal of LSC by increasing the expression of BCL2L2, a member of B cell lymphoma 2 (BCL2) family, by direct binding to its promoter region. Loss of WT1 impaired self-renewal ability in LSC and delayed the progression of leukemia. WP1130 was found to modify the WT1-BCL2L2 axis, and WP1130-induced anti-leukemic activity was mediated by ubiquitin proteasome-mediated destruction of WT1 protein. WP1130 induced apoptosis and decreased colony formation abilities of leukemia cells and prolonged the overall survival in the THP1-based xenograft NSG mouse model. WP1130 also decreased the frequency of LSC and prolonged the overall survival in MLL-AF9-induced murine leukemia model. Mechanistically, WP1130 induced the degradation of WT1 by positively affecting the ubiquitination of WT1 protein.
CONCLUSIONS: Our results indicate that WT1 is required for the development of AML. WP1130 exhibits anti-leukemic activity by inhibiting the WT1-BCL2L2 axis, which may represent a new acute myeloid leukemia therapy target.

Keywords:  Deubiquitinase inhibitor; Leukemia stem cell; Leukemia-initiating cell; Self-renewal; Ubiquitin–proteasome signal; Wilms’ tumor-1

DOI:  https://doi.org/10.1186/s12967-020-02384-y
Leukemia. 2020 Jun 22.

Bosutinib for pretreated patients with chronic phase chronic myeloid leukemia: primary results of the phase 4 BYOND study.

Andreas Hochhaus, Carlo Gambacorti-Passerini, Camille Abboud, Bjørn Tore Gjertsen, Tim H Brümmendorf, B Douglas Smith, Thomas Ernst, Pilar Giraldo-Castellano, Ulla Olsson-Strömberg, Susanne Saussele, Nathalie Bardy-Bouxin, Andrea Viqueira, Eric Leip, T Alexander Russell-Smith, Jocelyn Leone, Gianantonio Rosti, Justin Watts, Francis J Giles, .

Bosutinib is approved for newly diagnosed Philadelphia chromosome-positive (Ph+) chronic phase (CP) chronic myeloid leukemia (CML) and for Ph+ CP, accelerated (AP), or blast (BP) phase CML after prior treatment with tyrosine kinase inhibitors (TKIs). In the ongoing phase 4 BYOND study (NCT02228382), 163 CML patients resistant/intolerant to prior TKIs (n = 156 Ph+ CP CML, n = 4 Ph+ AP CML, n = 3 Ph-negative/BCR-ABL1+ CML) received bosutinib 500 mg once daily (starting dose). As of ≥1 year after last enrolled patient (median treatment duration 23.7 months), 56.4% of Ph+ CP CML patients remained on bosutinib. Primary endpoint of cumulative confirmed major cytogenetic response (MCyR) rate by 1 year was 75.8% in Ph+ CP CML patients after one or two prior TKIs and 62.2% after three prior TKIs. Cumulative complete cytogenetic response (CCyR) and major molecular response (MMR) rates by 1 year were 80.6% and 70.5%, respectively, in Ph+ CP CML patients overall. No patient progressed to AP/BP on treatment. Across all patients, the most common treatment-emergent adverse events were diarrhea (87.7%), nausea (39.9%), and vomiting (32.5%). The majority of patients had confirmed MCyR by 1 year and MMR by 1 year, further supporting bosutinib use for Ph+ CP CML patients resistant/intolerant to prior TKIs.

DOI: https://doi.org/10.1038/s41375-020-0915-9
Sci Rep. 2020 Jun 25. 10(1): 10325

Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia.

Hetty E Carraway, Sridhar A Malkaram, Yana Cen, Aymen Shatnawi, Jun Fan, Hamdy E A Ali, Zakaria Y Abd Elmageed, Thomm Buttolph, James Denvir, Donald A Primerano, Tamer E Fandy.

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

DOI: https://doi.org/10.1038/s41598-020-67170-8
Nat Commun. 2020 Jun 24. 11(1): 3194

Synergism between IL7R and CXCR4 drives BCR-ABL induced transformation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Hend Abdelrasoul, Anila Vadakumchery, Markus Werner, Lennart Lenk, Ahmad Khadour, Marc Young, Omar El Ayoubi, Fotini Vogiatzi, Markus Krämer, Vera Schmid, Zhengshan Chen, Yasar Yousafzai, Gunnar Cario, Martin Schrappe, Markus Müschen, Christina Halsey, Medhanie A Mulaw, Denis M Schewe, Elias Hobeika, Ameera Alsadeq, Hassan Jumaa.

Ph+ acute lymphoblastic leukemia (ALL) is characterized by the expression of an oncogenic fusion kinase termed BCR-ABL1. Here, we show that interleukin 7 receptor (IL7R) interacts with the chemokine receptor CXCR4 to recruit BCR-ABL1 and JAK kinases in close proximity. Treatment with BCR-ABL1 kinase inhibitors results in elevated expression of IL7R which enables the survival of transformed cells when IL7 was added together with the kinase inhibitors. Importantly, treatment with anti-IL7R antibodies prevents leukemia development in xenotransplantation models using patient-derived Ph+ ALL cells. Our results suggest that the association between IL7R and CXCR4 serves as molecular platform for BCR-ABL1-induced transformation and development of Ph+ ALL. Targeting this platform with anti-IL7R antibody eliminates Ph+ ALL cells including those with resistance to commonly used ABL1 kinase inhibitors. Thus, anti-IL7R antibodies may provide alternative treatment options for ALL in general and may suppress incurable drug-resistant leukemia forms.

DOI: https://doi.org/10.1038/s41467-020-16927-w
Cell Rep. 2020 Jun 23. pii: S2211-1247(20)30797-X. [Epub ahead of print]31(12): 107816

Targeting Bim via a lncRNA Morrbid Regulates the Survival of Preleukemic and Leukemic Cells.

Zhigang Cai, Fabiola Aguilera, Baskar Ramdas, Swapna Vidhur Daulatabad, Rajneesh Srivastava, Jonathan J Kotzin, Martin Carroll, Gerald Wertheim, Adam Williams, Sarath Chandra Janga, Chi Zhang, Jorge Henao-Mejia, Reuben Kapur.

  Inhibition of anti-apoptotic proteins BCL-2 and MCL-1 to release pro-apoptotic protein BIM and reactivate cell death could potentially be an efficient strategy for the treatment of leukemia. Here, we show that a lncRNA, MORRBID, a selective transcriptional repressor of BIM, is overexpressed in human acute myeloid leukemia (AML), which is associated with poor overall survival. In both human and animal models, MORRBID hyperactivation correlates with two recurrent AML drivers, TET2 and FLT3ITD. Mice with individual mutations of Tet2 or Flt3ITD develop features of chronic myelomonocytic leukemia (CMML) and myeloproliferative neoplasm (MPN), respectively, and combined presence results in AML. We observe increased levels of Morrbid in murine models of CMML, MPN, and AML. Functionally, loss of Morrbid in these models induces increased expression of Bim and cell death in immature and mature myeloid cells, which results in reduced infiltration of leukemic cells in tissues and prolongs the survival of AML mice.

Keywords:  BIM; FLT3(ITD); MORRBID; TET2; apoptosis; leukemia; lncRNA

DOI:  https://doi.org/10.1016/j.celrep.2020.107816
Blood Adv. 2020 Jun 23. 4(12): 2768-2778

Ex vivo drug screening defines novel drug sensitivity patterns for informing personalized therapy in myeloid neoplasms.

Michael A Spinner, Alexey Aleshin, Marianne T Santaguida, Steven A Schaffert, James L Zehnder, A Scott Patterson, Christos Gekas, Diane Heiser, Peter L Greenberg.

Precision medicine approaches such as ex vivo drug sensitivity screening (DSS) are appealing to inform rational drug selection in myelodysplastic syndromes (MDSs) and acute myeloid leukemia, given their marked biologic heterogeneity. We evaluated a novel, fully automated ex vivo DSS platform that uses high-throughput flow cytometry in 54 patients with newly diagnosed or treatment-refractory myeloid neoplasms to evaluate sensitivity (blast cytotoxicity and differentiation) to 74 US Food and Drug Administration-approved or investigational drugs and 36 drug combinations. After piloting the platform in 33 patients, we conducted a prospective feasibility study enrolling 21 patients refractory to hypomethylating agents (HMAs) to determine whether this assay could be performed within a clinically actionable time frame and could accurately predict clinical responses in vivo. When assayed for cytotoxicity, ex vivo drug sensitivity patterns were heterogeneous, but they defined distinct patient clusters with differential sensitivity to HMAs, anthracyclines, histone deacetylase inhibitors, and kinase inhibitors (P < .001 among clusters) and demonstrated synergy between HMAs and venetoclax (P < .01 for combinations vs single agents). In our feasibility study, ex vivo DSS results were available at a median of 15 days after bone marrow biopsy, and they informed personalized therapy, which frequently included venetoclax combinations, kinase inhibitors, differentiative agents, and androgens. In 21 patients with available ex vivo and in vivo clinical response data, the DSS platform had a positive predictive value of 0.92, negative predictive value of 0.82, and overall accuracy of 0.85. These data demonstrate the utility of this approach for identifying potentially useful and often novel therapeutic drugs for patients with myeloid neoplasms refractory to standard therapies.

DOI: https://doi.org/10.1182/bloodadvances.2020001934
Blood. 2020 Jun 24. pii: blood.2020005827. [Epub ahead of print]

Lysine-specific demethylase 1A (LSD1) restricts ex vivo propagation of human HSCs and is a target of UM171.

Agatheeswaran Subramaniam, Kristijonas Žemaitis, Mehrnaz Safaee Talkhoncheh, David Yudovich, Alexandra Bäckström, Shubhranshu Debnath, Jun Chen, Mayur Vilas Jain, Roman Galeev, Massimiliano Gaetani, Roman A Zubarev, Jonas Larsson.

Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. Here, we report that inhibition of the epigenetic regulator Lysine-specific histone demethylase 1A (LSD1) induces a rapid expansion of human cord blood derived CD34+ cells and promotes in vitro propagation of long-term repopulating HSCs by preventing differentiation. The phenotype and molecular characteristics of cells treated with LSD1 inhibitors were highly similar to cells treated with UM171, an agent promoting expansion of HSCs through undefined mechanisms, and currently tested in clinical trials. Strikingly, we found that LSD1 as well as other members of the LSD1 containing chromatin remodeling complex CoREST are rapidly poly-ubiquitinated and degraded upon UM171 treatment. CRISPR/Cas9 depletion of the CoREST core member, RCOR1, resulted in expansion of CD34+ cells similar to LSD1 inhibition and UM171. Taken together, LSD1 and CoREST restrict HSC expansion, and are principal targets of UM171, forming a mechanistic basis for the HSC promoting activity of UM171.

DOI: https://doi.org/10.1182/blood.2020005827
Nature. 2020 Jun 24.

FLT3 stop mutation increases FLT3 ligand level and risk of autoimmune thyroid disease.

Saedis Saevarsdottir, Thorunn A Olafsdottir, Erna V Ivarsdottir, Gisli H Halldorsson, Kristbjorg Gunnarsdottir, Asgeir Sigurdsson, Ari Johannesson, Jon K Sigurdsson, Thorhildur Juliusdottir, Sigrun H Lund, Asgeir O Arnthorsson, Edda L Styrmisdottir, Julius Gudmundsson, Gerdur M Grondal, Kristjan Steinsson, Lars Alfredsson, Johan Askling, Rafn Benediktsson, Ragnar Bjarnason, Arni J Geirsson, Bjorn Gudbjornsson, Hallgrimur Gudjonsson, Haukur Hjaltason, Astradur B Hreidarsson, Lars Klareskog, Ingrid Kockum, Helga Kristjansdottir, Thorvardur J Love, Bjorn R Ludviksson, Tomas Olsson, Pall T Onundarson, Kjartan B Orvar, Leonid Padyukov, Bardur Sigurgeirsson, Vinicius Tragante, Kristbjorg Bjarnadottir, Thorunn Rafnar, Gisli Masson, Patrick Sulem, Daniel F Gudbjartsson, Pall Melsted, Gudmar Thorleifsson, Gudmundur L Norddahl, Unnur Thorsteinsdottir, Ingileif Jonsdottir, Kari Stefansson.

Autoimmune thyroid disease is the most common autoimmune disease and is highly heritable1. Here, by using a genome-wide association study of 30,234 cases and 725,172 controls from Iceland and the UK Biobank, we find 99 sequence variants at 93 loci, of which 84 variants are previously unreported2-7. A low-frequency (1.36%) intronic variant in FLT3 (rs76428106-C) has the largest effect on risk of autoimmune thyroid disease (odds ratio (OR) = 1.46, P = 2.37 × 10-24). rs76428106-C is also associated with systemic lupus erythematosus (OR = 1.90, P = 6.46 × 10-4), rheumatoid factor and/or anti-CCP-positive rheumatoid arthritis (OR = 1.41, P = 4.31 × 10-4) and coeliac disease (OR = 1.62, P = 1.20 × 10-4). FLT3 encodes fms-related tyrosine kinase 3, a receptor that regulates haematopoietic progenitor and dendritic cells. RNA sequencing revealed that rs76428106-C generates a cryptic splice site, which introduces a stop codon in 30% of transcripts that are predicted to encode a truncated protein, which lacks its tyrosine kinase domains. Each copy of rs76428106-C doubles the plasma levels of the FTL3 ligand. Activating somatic mutations in FLT3 are associated with acute myeloid leukaemia8 with a poor prognosis and rs76428106-C also predisposes individuals to acute myeloid leukaemia (OR = 1.90, P = 5.40 × 10-3). Thus, a predicted loss-of-function germline mutation in FLT3 causes a reduction in full-length FLT3, with a compensatory increase in the levels of its ligand and an increased disease risk, similar to that of a gain-of-function mutation.

DOI: https://doi.org/10.1038/s41586-020-2436-0
Br J Haematol. 2020 Jun 25.

The loss or absence of minimal residual disease of <0·1% at any time after two cycles of consolidation chemotherapy in CBFB-MYH11-positive acute myeloid leukaemia indicates poor prognosis.

Wenbing Duan, Xiaohong Liu, Jinsong Jia, Jing Wang, Lizhong Gong, Qian Jiang, Ting Zhao, Yu Wang, Xiaohui Zhang, Lanping Xu, Xiaosu Zhao, Yazhen Qin, Hongxia Shi, Yingjun Chang, Xiaojun Huang, Hao Jiang.

  No consensus has been reached on the relationship between CBFB-MYH11 copies and prognosis. Of 1525 acute myeloid leukemia (AML) patients, 58 with CBFB-MYH11-positive AML (16/58 patients with c-kit mutation) were retrospectively analyzed with a median follow-up duration of 29.8 (range: 4.8-74.4) months. Of these, 25/58 (43.1%) patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), 10 of whom had the c-kit mutation. Of the 33 patients who did not undergo allo-HSCT, recurrence in patients with CBFB-MYH11/ABL level >0.1% at any time after two consolidation cycles was significantly higher than in patients with CBFB-MYH11/ABL level <0.1% (61.9% vs. 0%, P = 0.001); further, the 3-year relapse-free survival (RFS; 31.4% vs. 100%, P = 0.004) and event-free survival (EFS; 33.1% vs. 100%, P = 0.004) were significantly decreased in patients with CBFB-MYH11/ABL level >0.1% at any time after two consolidation cycles. The 3-year RFS and EFS rates were lower in patients who did not receive allo-HSCT than in those who did (31.4% vs 84.6%, P = 0.000; 31.4% vs. 80.8%, P = 0.001). CBFB-MYH11-positive AML patients with CBFB-MYH11/ABL level >0.1% at any time after two cycles of consolidation had poor prognoses, and allo-HSCT could improve their survival.

Keywords:   CBFB-MYH11 ; acute myeloid leukaemia; consolidation; minimal residual disease; prognosis

DOI:  https://doi.org/10.1111/bjh.16745
Leukemia. 2020 Jun 24.

Clonal dominance is an adverse prognostic factor in acute myeloid leukemia treated with intensive chemotherapy.

Marco Cerrano, Matthieu Duchmann, Rathana Kim, Loic Vasseur, Pierre Hirsch, Xavier Thomas, Samuel Quentin, Justine Pasanisi, Marie Passet, Florence Rabian, Ramy Rahmé, Etienne Lengliné, Emmanuel Raffoux, Nathalie Dhédin, Marie Sébert, Odile Maarek, Anna Raimbault, Karine Celli-Lebras, Lionel Adès, Pierre Fenaux, Nicolas Boissel, François Delhommeau, Jean Soulier, Hervé Dombret, Emmanuelle Clappier, Pierre Sujobert, Raphael Itzykson.

Intra-tumor heterogeneity portends poor outcome in many cancers. In AML, a higher number of drivers worsens prognosis. The Shannon Index is a robust metric of clonal heterogeneity that accounts for the number of clones, but also their relative abundance. We show that a Shannon Index can be estimated from bulk sequencing, which is correlated (ρ = 0.76) with clonal diversity from single-colony genotyping. In a discovery cohort of 292 patients with sequencing of 43 genes, a higher number of drivers (HR = 1.18, P = 0.028) and a lower Shannon Index (HR = 0.68, P = 0.048), the latter reflecting clonal dominance, are independently associated with worse OS independently of European LeukemiaNet 2017 risk. These findings are validated in an independent cohort of 1184 patients with 111-gene sequencing (number of drivers HR = 1.16, P = 1 × 10-5, Shannon Index HR = 0.81, P = 0.007). By re-interrogating paired diagnosis/relapse exomes from 50 cytogenetically normal AMLs, we find clonal dominance at diagnosis to be correlated with the gain of a significantly higher number of mutations at relapse (P = 6 × 10-6), hence with clonal sweeping. Our results suggest that clonal dominance at diagnosis is associated with the presence of a leukemic phenotype allowing rapid expansion of new clones and driving relapse after chemotherapy.

DOI: https://doi.org/10.1038/s41375-020-0932-8
Oncogene. 2020 Jun 22.

Different impact of calreticulin mutations on human hematopoiesis in myeloproliferative neoplasms.

Mira El-Khoury, Xénia Cabagnols, Matthieu Mosca, Gaëlle Vertenoeil, Christophe Marzac, Fabrizia Favale, Olivier Bluteau, Florence Lorre, Amandine Tisserand, Graciela Rabadan Moraes, Valérie Ugo, Jean-Christophe Ianotto, Jerôme Rey, Eric Solary, Lydia Roy, Philippe Rameau, Najet Debili, Florence Pasquier, Nicole Casadevall, Caroline Marty, Stefan N Constantinescu, Hana Raslova, William Vainchenker, Isabelle Plo.

Mutations of calreticulin (CALRm) define a subtype of myeloproliferative neoplasms (MPN). We studied the biological and genetic features of CALR-mutated essential thrombocythemia and myelofibrosis patients. In most cases, CALRm were found in granulocytes, monocytes, B and NK cells, but also in T cells. However, the type 1 CALRm spreads more easily than the type 2 CALRm in lymphoid cells. The CALRm were also associated with an early clonal dominance at the level of hematopoietic stem and progenitor cells (HSPC) with no significant increase during granulo/monocytic differentiation in most cases. Moreover, we found that half of type 2 CALRm patients harbors some homozygous progenitors. Those patients were associated with a higher clonal dominance during granulo/monocytic differentiation than patients with only heterozygous type 2 CALRm progenitors. When associated mutations were present, CALRm were the first genetic event suggesting that they are both the initiating and phenotypic event. In blood, type 1 CALRm led to a greater increased number of all types of progenitors compared with the type 2 CALRm. However, both types of CALRm induced an increase in megakaryocytic progenitors associated with a ruxolitinib-sensitive independent growth and with a mild constitutive signaling in megakaryocytes. At the transcriptional level, type 1 CALRm seems to deregulate more pathways than the type 2 CALRm in megakaryocytes. Altogether, our results show that CALRm modify both the HSPC and megakaryocyte biology with a stronger effect for type 1 than for type 2 CALRm.

DOI: https://doi.org/10.1038/s41388-020-1368-3
Cancer Cell. 2020 Jun 09. pii: S1535-6108(20)30268-3. [Epub ahead of print]

PKCλ/ι Loss Induces Autophagy, Oxidative Phosphorylation, and NRF2 to Promote Liver Cancer Progression.

Yotaro Kudo, Masayuki Sugimoto, Esperanza Arias, Hiroaki Kasashima, Thekla Cordes, Juan F Linares, Angeles Duran, Yuki Nakanishi, Naoko Nakanishi, Antoine L'Hermitte, Alex Campos, Nadia Senni, Tarmo Rooslid, Lewis R Roberts, Ana Maria Cuervo, Christian M Metallo, Michael Karin, Maria T Diaz-Meco, Jorge Moscat.

  Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during reactive oxygen species (ROS) generation, and how ROS promote tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) λ/ι loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC through cell-autonomous and non-autonomous mechanisms. Although PKCλ/ι promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKCλ/ι levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.

Keywords:  NRF2; PKCζ; PKCι; PKCλ; atypical PKC; autophagy; hepatocellular carcinoma; metabolic reprogramming; oxidative phosphorylation; reactive oxygen species

DOI:  https://doi.org/10.1016/j.ccell.2020.05.018
Leukemia. 2020 Jun 24.

CARM1 inhibition reduces histone acetyltransferase activity causing synthetic lethality in CREBBP/EP300-mutated lymphomas.

Kylee J Veazey, Donghang Cheng, Kevin Lin, Oscar D Villarreal, Guozhen Gao, Mabel Perez-Oquendo, Hieu T Van, Sabrina A Stratton, Michael Green, Han Xu, Yue Lu, Mark T Bedford, Margarida Almeida Santos.

Somatic mutations affecting CREBBP and EP300 are a hallmark of diffuse large B-cell lymphoma (DLBCL). These mutations are frequently monoallelic, within the histone acetyltransferase (HAT) domain and usually mutually exclusive, suggesting that they might affect a common pathway, and their residual WT expression is required for cell survival. Using in vitro and in vivo models, we found that inhibition of CARM1 activity (CARM1i) slows DLBCL growth, and that the levels of sensitivity are positively correlated with the CREBBP/EP300 mutation load. Conversely, treatment of DLBCLs that do not have CREBBP/EP300 mutations with CARM1i and a CBP/p300 inhibitor revealed a strong synergistic effect. Our mechanistic data show that CARM1i further reduces the HAT activity of CBP genome wide and downregulates CBP-target genes in DLBCL cells, resulting in a synthetic lethality that leverages the mutational status of CREBBP/EP300 as a biomarker for the use of small-molecule inhibitors of CARM1 in DLBCL and other cancers.

DOI: https://doi.org/10.1038/s41375-020-0908-8
Nature. 2020 Jun 24.

Pervasive lesion segregation shapes cancer genome evolution.

Sarah J Aitken, Craig J Anderson, Frances Connor, Oriol Pich, Vasavi Sundaram, Christine Feig, Tim F Rayner, Margus Lukk, Stuart Aitken, Juliet Luft, Elissavet Kentepozidou, Claudia Arnedo-Pac, Sjoerd V Beentjes, Susan E Davies, Ruben M Drews, Ailith Ewing, Vera B Kaiser, Ava Khamseh, Erika López-Arribillaga, Aisling M Redmond, Javier Santoyo-Lopez, Inés Sentís, Lana Talmane, Andrew D Yates, , Colin A Semple, Núria López-Bigas, Paul Flicek, Duncan T Odom, Martin S Taylor.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion1,2. Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

DOI: https://doi.org/10.1038/s41586-020-2435-1