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Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide definitive evidence for Club cells as progenitors for LUAD. Using in vivo lineage tracing, we find that a subset of Kras12V -expressing and Trp53-deficient Club cells act as precursors for LUAD and we define the stepwise trajectory of Club cell-initiated tumors leading to lineage marker conversion and aggressive LUAD. Our results establish Club cells as cells-of-origin for LUAD and demonstrate that Club cell-initiated tumors have the potential to develop aggressive LUAD.

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03174-1.

Background Acute myeloid leukemia (AML) is a heterogeneous disease of the hematopoietic progenitor cell driven by the subsequent acquisition of genetic alterations. Approximately 20% of AML patients show strong expression of CD56 (neural cell adhesion molecule; NCAM). Expression of NCAM is associated with poor overall survival; however, the functional role of aberrant NCAM expression has not been investigated to date. The goal of this study is to examine the biological role of NCAM in AML and to explore whether NCAM represents a potential therapeutic target. Results In order to evaluate the clinical significance of elevated NCAM expression in AML, we screened a panel of human cell lines for CD56 expression. Most cell lines were positive and cell surface expression correlated with mRNA levels. Knockdown of NCAM with three different doxycycline-inducible shRNAs suppressed cell growth and MTT activity in all positive cell lines. Propidium iodide staining demonstrated an increase in G1 arrest. Importantly, there was a marked apoptosis after NCAM suppression and this effect was proportional to the knockdown efficiency. Survival of NOD-SCIDgamma chain mice in a leukemia engraftment model was significantly prolonged upon NCAM knockdown. Suppression of NCAM sensitized leukemic blasts to treatment with Ara-C or Daunorubicin in vitro and in xenotransplantation experiments. To test the consequences of NCAM overexpression in negative leukemic cell lines we transduced the NCAM transcript from Nomo-1 into HL60 and K562 cells. HL60 cells had a significantly lower sensitivity towards Ara-C or Daunorubicin treatment. IC50 for the BCR-ABL inhibitor Dasatinib in K562 cells increased from 0.95 nM (EV, empty vector) to 2.2 nM in NCAM overexpressing cells. To dissect possible upstream regulation mechanisms of NCAM expression we performed DNAseI hypersensitivity assays coupled to qRT-PCR mapping of known putative sites in the NCAM promoter and observed open chromatin for the binding sites of Meis1, Mef2 and Stat1. shRNA-mediated knockdown of MEIS1, MEF2c and MLL-AF9 resulted in significant suppression of NCAM cell surface expression, suggesting an upstream regulatory role for MLL-AF9. To gain insights into the mechanisms underlying the NCAM function in AML we performed gene expression comparisons of the 30 highest versus 30 lowest expressing samples in the GSE8043 dataset. Fifty-seven Biocharta pathways were differentially expressed between NCAMhigh and NCAMlow samples, while expression changes predicted abnormal cell-cycle regulation, stress and DNA damage response, cell survival, renewal and adhesion. Western blot, protein array and qRT-PCR analyses of candidate downstream signaling pathways upon knockdown of NCAM demonstrated enhanced degradation of BetaCatenin, decreased expression of BCL-2 and increased levels of p21 and p27. The upstream regulation mechanism described above revealed MLL-AF9 (M/A9) as a top candidate for NCAM regulation. Subsequent analysis of M/A9 L-GMPs (Lin- cKit+ CD34+ FcgR+) demonstrated strong surface expression of NCAM, whereas normal HSCs (Lin-cKit+ Sca1+) were NCAM-negative. This could be validated by gene expression analyses of M/A9 L-GMPs compared with normal HSCs. In order to elucidate the role of NCAM on leukemic cell function in a mouse model, NCAM-/- and control wildtype (WT) bone marrow cells were transformed with a retroviral construct of M/A9 and transplanted into lethally irradiated littermates. Recipients of NCAM-/- M/A9 cells developed acute leukemia with prolonged disease latency. NCAM-/- M/A9 cells had lower CD117 and Gr-1 expression, but higher expression of Mac-1 and, in some samples, aberrant B220 co-expression. Importantly, there was a reduced representation of L-GMPs in the NCAM-/- M/A9 group and limited dilution retransplantation assays revealed a significantly prolonged survival of NCAM-/- M/A9 mice. Replating activity in methylcellulose was diminished and could be eradicated with sublethal doses of Cytarabine. Summary Targeting aberrant expression of NCAM demonstrated strong antileukemic activity in vitro and sensitized leukemic blasts to genotoxic stress. In vivo, depletion of NCAM resulted in prolonged disease survival in syngeneic and xenotransplantation experiments and diminished self-renewal capacities. Our data suggest that NCAM represents a promising therapeutic strategy and likely targets AML cells at the LSC level. Disclosures No relevant conflicts of interest to declare.

Background SIRT1 is a NAD+ dependent histone deacetylase, which has been shown to act as an important regulator of apoptosis, DNA-repair and is involved in the maintenance of genetic integrity under conditions of cellular stress. Beside deacetylation of histones H4K16, SIRT1 has numeral other substrates including KU70, FOXO1 or p53. SIRT1 deacetylates p53 at lysine 382 thereby reducing its transcriptional activity followed by loss of p53 dependent apoptosis in response to cell damage. The activity of SIRT1 is negatively regulated by DBC1 (Deleted in Breast Cancer 1) and involves protein–protein interaction (Kim et al., Nature 2008). Recent reports have demonstrated increased expression of SIRT1 in leukemic cells. Of note, SIRT1 seems to be specifically essential to maintain stem cell activity in CML leukemic stem cells (Li et al, Cancer Cell, 2012), however the role of SIRT1 in acute myeloid leukemia (AML) and in the context of other oncogenic tyrosine kinases is poorly understood. Aims To characterize the role of SIRT1 in acute myeloid leukemia in the context of defined genetic backgrounds. Methods To investigate the functional role of SIRT1 we performed expression analysis studies. Inhibition of SIRT1 was achieved by shRNA experiments and pharmacological targeting. Functional consequences were addressed by immunoblotting, co-immunoprecipitation, cell death assays, gamma-H2AX staining and xenotransplantion experiments. Results SIRT1 protein expression analysis in primary human AML samples (n=20) and cell lines (n=13) showed preferential high expression levels in cells harboring FLT3-ITD mutations or oncogenic KRAS/NRAS. Inhibition of FLT3 signaling using shRNA-mediated knockdown of FLT3 or upon treatment with tyrosine kinase inhibitors caused a time and dose dependent suppression of SIRT1 expression accompanied by an increase in p53 acetylation. Vice versa, overexpression of FLT3-ITD in BaF/3 cells or in several murine leukemia models (e.g. MLL/AF9 +/-FLT3-ITD; AML1/ETO +/-FLT3-ITD) induced significant upregulation of SIRT1. Targeting SIRT1 using the SIRT1/2 inhibitor Tenovin-6 (TV-6) or knockdown of SIRT1 resulted in a slight increase in apoptotic cell death in primary AML samples and cell lines. In contrast, inhibition of SIRT1 significantly sensitized to FLT3 inhibitor therapy. Of note, the combination of TV-6 and midostaurin inhibited colony growth and replating activity in MLL-AF9 or AML1-ETO cells harboring FLT3-ITD mutations. Further, knockdown of SIRT1 in the AML cell line MV4-11 expressing a doxycycline-regulated SIRT-shRNA resulted in prolonged survival in a xenotransplantation model. The observed effects prompted us to investigate whether mutated FLT3 is directly involved in the regulation of SIRT1. Inhibition of FLT3-ITD kinase activity caused an increased phosphorylation of the physiological SIRT1-inhibitor DBC1 followed by binding of SIRT1 as revealed by co-immunoprecipitation experiments. We also noticed a slight increase in ATM/ATR phosphorylation and an increase in ATM-DBC1 binding upon FLT3-ITD inhibition indicating SIRT1 inhibition via the ATM-DBC1-SIRT1 axis. These effects were substantially enhanced upon treatment with genotoxic agents (cytarabine; irradiation) in combination with midostaurin compared to either agent alone and were accompanied by increased p53 acetylation and an increased number of gamma-H2AX foci. Conclusion SIRT1 is overexpressed in AML cells and partially regulated by FLT3-ITD or oncogenic RAS. In FLT3-ITD mutated cells SIRT1 is constitutively activated, likely via the inhibition of the physiological inhibitor DBC1. Pharmacologic targeting of SIRT1 sensitizes AML cells to TKI therapy or genotoxic agents and partially restored the FLT3-ITD associated defective stress response pathway ATM-DBC1-SIRT1 resulting in activation of p53. Disclosures: Kindler: Novartis: Membership on an entity’s Board of Directors or advisory committees.