Comparison of acute myeloid leukemia and myelodysplastic syndromes with TP53 aberrations.

TP53 aberrations are found in approximately 10% of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and are considered early driver events affecting leukemia stem cells. In this study, we compared features of a total of 84 patients with these disorders seen at a tertiary cancer center. Clinical and cytogenetic characteristics as well as immunophenotypes of immature blast cells were similar between AML and MDS patients. Median overall survival (OS) was 226 days (95% confidence interval [CI], 131-300) for the entire cohort with an estimated 3-year OS rate of 11% (95% CI, 6-22). OS showed a significant difference between MDS (median, 345 days; 95% CI, 235-590) and AML patients (median, 91 days; 95% CI, 64-226) which is likely due to a different co-mutational pattern as revealed by next-generation sequencing. Transformation of TP53 aberrant MDS occurred in 60.5% of cases and substantially reduced their survival probability. Cox regression analysis revealed treatment class and TP53 variant allele frequency as prognostically relevant parameters but not the TP53-specific prognostic scores EAp53 and RFS. These data emphasize similarities between TP53 aberrant AML and MDS and support previous notions that they should be classified and treated as a distinct disorder.

A novel function of STAT3ß in suppressing interferon response improves outcome in acute myeloid leukemia.

Signal transducer and activator of transcription 3 (STAT3) is frequently overexpressed in patients with acute myeloid leukemia (AML). STAT3 exists in two distinct alternatively spliced isoforms, the full-length isoform STAT3α and the C-terminally truncated isoform STAT3ß. While STAT3α is predominantly described as an oncogenic driver, STAT3ß has been suggested to act as a tumor suppressor. To elucidate the role of STAT3ß in AML, we established a mouse model of STAT3ß-deficient, MLL-AF9-driven AML. STAT3ß deficiency significantly shortened survival of leukemic mice confirming its role as a tumor suppressor. Furthermore, RNA sequencing revealed enhanced STAT1 expression and interferon (IFN) signaling upon loss of STAT3ß. Accordingly, STAT3ß-deficient leukemia cells displayed enhanced sensitivity to blockade of IFN signaling through both an IFNAR1 blocking antibody and the JAK1/2 inhibitor Ruxolitinib. Analysis of human AML patient samples confirmed that elevated expression of IFN-inducible genes correlated with poor overall survival and low STAT3ß expression. Together, our data corroborate the tumor suppressive role of STAT3ß in a mouse model in vivo. Moreover, they provide evidence that its tumor suppressive function is linked to repression of the STAT1-mediated IFN response. These findings suggest that the STAT3ß/α mRNA ratio is a significant prognostic marker in AML and holds crucial information for targeted treatment approaches. Patients displaying a low STAT3ß/α mRNA ratio and unfavorable prognosis could benefit from therapeutic interventions directed at STAT1/IFN signaling.

STAT3 in acute myeloid leukemia facilitates natural killer cell-mediated surveillance.

Acute myeloid leukemia (AML) is a heterogenous disease characterized by the clonal expansion of myeloid progenitor cells. Despite recent advancements in the treatment of AML, relapse still remains a significant challenge, necessitating the development of innovative therapies to eliminate minimal residual disease. One promising approach to address these unmet clinical needs is natural killer (NK) cell immunotherapy. To implement such treatments effectively, it is vital to comprehend how AML cells escape the NK-cell surveillance. Signal transducer and activator of transcription 3 (STAT3), a component of the Janus kinase (JAK)-STAT signaling pathway, is well-known for its role in driving immune evasion in various cancer types. Nevertheless, the specific function of STAT3 in AML cell escape from NK cells has not been deeply investigated. In this study, we unravel a novel role of STAT3 in sensitizing AML cells to NK-cell surveillance. We demonstrate that STAT3-deficient AML cell lines are inefficiently eliminated by NK cells. Mechanistically, AML cells lacking STAT3 fail to form an immune synapse as efficiently as their wild-type counterparts due to significantly reduced surface expression of intercellular adhesion molecule 1 (ICAM-1). The impaired killing of STAT3-deficient cells can be rescued by ICAM-1 overexpression proving its central role in the observed phenotype. Importantly, analysis of our AML patient cohort revealed a positive correlation between ICAM1 and STAT3 expression suggesting a predominant role of STAT3 in ICAM-1 regulation in this disease. In line, high ICAM1 expression correlates with better survival of AML patients underscoring the translational relevance of our findings. Taken together, our data unveil a novel role of STAT3 in preventing AML cells from escaping NK-cell surveillance and highlight the STAT3/ICAM-1 axis as a potential biomarker for NK-cell therapies in AML.

Inability to phosphorylate Y88 of p27Kip1 enforces reduced p27 protein levels and accelerates leukemia progression.

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 regulates cell proliferation. Phosphorylation of tyrosine residue 88 (Y88) converts the inhibitor into an assembly factor and activator of CDKs, since Y88-phosphorylation restores activity to cyclin E,A/CDK2 and enables assembly of active cyclin D/CDK4,6. To investigate the physiological significance of p27 tyrosine phosphorylation, we have generated a knock-in mouse model where Y88 was replaced by phenylalanine (p27-Y88F). Young p27-Y88F mice developed a moderately reduced body weight, indicative for robust CDK inhibition by p27-Y88F. When transformed with v-ABL or BCR::ABL1p190, primary p27-Y88F cells are refractory to initial transformation as evidenced by a diminished outgrowth of progenitor B-cell colonies. This indicates that p27-Y88 phosphorylation contributes to v-ABL and BCR::ABL1p190 induced transformation. Surprisingly, p27-Y88F mice succumbed to premature v-ABL induced leukemia/lymphoma compared to p27 wild type animals. This was accompanied by a robust reduction of p27-Y88F levels in v-ABL transformed cells. Reduced p27-Y88F levels seem to be required for efficient cell proliferation and may subsequently support accelerated leukemia progression. The potent downregulation p27-Y88F levels in all leukemia-derived cells could uncover a novel mechanism in human oncogenesis, where reduced p27 levels are frequently observed.

Specific effects of somatic GATA2 zinc finger mutations on erythroid differentiation.

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34+ bone marrow cells and in the myeloid cell line K562, ectopic expression of GATA2 L359V, but not A318T or G320D, caused a block of erythroid differentiation accompanied by downregulation of GATA1, STAT5B, and PLCG1. Our findings may explain the role of GATA2 L359V during the progression of chronic myeloid leukemia and the collaboration of GATA2 ZF1 alterations with CEBPA double mutations in erythroleukemia.

Functional Classification of TP53 Mutations in Acute Myeloid Leukemia.

Mutations of the TP53 gene occur in a subset of patients with acute myeloid leukemia (AML) and confer an exceedingly adverse prognosis. However, whether different types of TP53 mutations exert a uniformly poor outcome has not been investigated yet. Here, we addressed this issue by analyzing data of 1537 patients intensively treated within protocols of the German-Austrian AML study group. We classified TP53 mutations depending on their impact on protein structure and according to the evolutionary action (EAp53) score and the relative fitness score (RFS). In 98/1537 (6.4%) patients, 108 TP53 mutations were detected. While the discrimination depending on the protein structure and the EAp53 score did not show a survival difference, patients with low-risk and high-risk AML-specific RFS showed a different overall survival (OS; median, 12.9 versus 5.5 months, p = 0.017) and event-free survival (EFS; median, 7.3 versus 5.2 months, p = 0.054). In multivariable analyses adjusting for age, gender, white blood cell count, cytogenetic risk, type of AML, and TP53 variant allele frequency, these differences were statistically significant for both OS (HR, 2.14; 95% CI, 1.15-4.0; p = 0.017) and EFS (HR, 1.97; 95% CI, 1.06-3.69; p = 0.033). We conclude that the AML-specific RFS is of prognostic value in patients with TP53-mutated AML and a useful tool for therapeutic decision-making.

Acute myeloid leukemia driven by the CALM-AF10 fusion gene is dependent on BMI1.

A subset of acute myeloid and lymphoid leukemia cases harbor a t(10;11)(p13;q14) translocation resulting in the CALM-AF10 fusion gene. Standard chemotherapeutic strategies are often ineffective in treating patients with CALM-AF10 fusions. Hence, there is an urgent need to identify molecular pathways dysregulated in CALM-AF10-positive leukemias which may lay the foundation for novel targeted therapies. Here we demonstrate that the Polycomb Repressive Complex 1 gene BMI1 is consistently overexpressed in adult and pediatric CALM-AF10-positive leukemias. We demonstrate that genetic Bmi1 depletion abrogates CALM-AF10-mediated transformation of murine hematopoietic stem and progenitor cells (HSPCs). Furthermore, CALM-AF10-positive murine and human AML cells are sensitive to the small-molecule BMI1 inhibitor PTC-209 as well as to PTC-596, a compound in clinical development that has been shown to result in downstream degradation of BMI1 protein. PTC-596 significantly prolongs survival of mice injected with a human CALM-AF10 cell line in a xenograft assay. In summary, these results validate BMI1 as a bona fide candidate for therapeutic targeting in AML with CALM-AF10 rearrangements.

ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation.

The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.

Identifying novel mycobacterial stress associated genes using a random mutagenesis screen in Mycobacterium smegmatis.

Cell envelope associated components of Mycobacterium tuberculosis (M.tb) have been implicated in stress response, immune modulation and in vivo survival of the pathogen. Although many such factors have been identified, there is a large disparity between the number of genes predicted to be involved in functions linked to the envelope and those described in the literature. To identify and characterise novel stress related factors associated with the mycobacterial cell envelope, we isolated colony morphotype mutants of Mycobacterium smegmatis (M. smegmatis), based on the hypothesis that mutants with unusual colony morphology may have defects in the biosynthesis of cell envelope components. On testing their susceptibility to stress conditions relevant to M.tb physiology, multiple mutants were found to be sensitive to Isoniazid, Diamide and H2O2, indicative of altered permeability due to changes in cell envelope composition. Two mutants showed defects in biofilm formation implying possible roles for the target genes in antibiotic tolerance and/or virulence. These assays identified novel stress associated roles for several mycobacterial genes including sahH, tatB and aceE. Complementation analysis of selected mutants with the M. smegmatis genes and their M.tb homologues showed phenotypic restoration, validating their link to the observed phenotypes. A mutant carrying an insertion in fhaA encoding a forkhead associated domain containing protein, showed reduced survival in THP-1 macrophages, providing in vivo validation to this screen. Taken together, these results suggest that the M.tb homologues of a majority of the identified genes may play significant roles in the pathogenesis of tuberculosis.