Periodic Reporting for period 3 - MAMLE (Understanding the mechanisms of human acute myeloid leukaemia (AML) evolution)
Reporting period: 2020-02-01 to 2021-07-31
In this study we attempted to answer some of these questions. We sequenced genes related to AML from the peripheral blood (PB) of 124 individuals with AML from the EPIC project. Blood was available, on average, 7 years prior to AML diagnosis. Results were compared to 681 controls. In brief, we found that compared to healthy controls, in pre-AML cases more mutations were found, with larger clones based on variant allele frequency (VAF), in specific positions of the genome which are recurrently mutated in patients with hematologic malignancies. Most strikingly, spliceosome machinery mutations (SMM) were almost exclusively found in pre-AML cases and tended to arise at an earlier age when compared to individuals who carry them but do not proceed to develop the disease. Collectively, our findings provide new insights into the pre-clinical evolution of AML and support the hypothesis that individuals at high risk of AML development can be identified years before they develop overt disease.
Another main problem is that many AML patients will relapse after initial successful therapy and succumb to their disease. In our studies, we showed that the origins of AML relapse are heterogeneous but converge to stem cell properties of the relapsing clones In order to prevent relapse, leukemic stem cells should be eradicated in early stages of treatment (induction/remission) before they expand and acquire additional resistance mechanisms. It has been suggested that combination therapy which targets several phenotypes of the leukemic cells could be useful in overcoming AML heterogeneity. AML, which is mainly a disease of the elderly, the older individuals are less tolerable to the additive side effects of combination therapy. Therefore applying the right drug with minimal toxicity, the right set of patients, might be a valid approach. In this study, our global aim was to identify therapies and biomarkers, that can predict which drugs might be added to induction therapy to prevent relapse in specific subtypes of AML. Here, we studied patterns of in vitro drug response, which correlates with molecular characteristics of primary AML patients who achieved Complete Remission. In the current study we discovered that samples sensitive in vitro to dasatinib (a multi kinase inhibitor) had a specific in vitro drug sensitivity pattern, gene expression signature, and were enriched in FLT3/ITD and PTPN11 mutations. Dasatinib demonstrated anti LSC effect in vivo and the response to dasatinib among carriers of FLT3/ITD could be predicted based on gene expression of dasatinib targets.
2. To identify ARCH mutations with the high probability of AML transformation, we compared the mutational landscape of ARCH in two different groups: 95 individuals who later developed AML with a median of 6.3 years before AML diagnosis (pre-AML group) and 414 age/gender-matched individuals who did not develop AML with an average of 11.6 years follow-up (control group) (Fig 1). Error-corrected deep sequencing of recurrently mutated genes in AML showed that the pre-AML group had significantly higher prevalence of ARCH, higher variant allele frequency (VAF) of detected ARCH mutations, and a higher number of ARCH mutations per individual compared to the control group. We then calculated the relative risk of individual ARCH mutation for AML development and validated the results in a validation cohort. This analysis revealed a remarkable heterogeneity of AML risk among ARCH mutations and identified TP53 (hazard ratio [HR] = 12.5 95% CI: 5.0–160.5) and U2AF1 (HR = 7.9 95% CI: 4.1–192.2) mutations as having the highest risk for AML development, while the risk was relatively small in DNMT3A mutations (Fig 2). Additionally, the presence of two or more ARCH mutations per individual and ARCH mutations with VAF ≥ 9% were also associated with a high risk of AML transformation. (Abelson S,…..Shlush L,. 2018. Nature 559, pages400–404)
3. We studied patterns of in vitro drug response among primary AML patients who achieved complete remission (CR). Results from the current study were compared to the Beat AML study. We performed unsupervised hierarchical clustering analysis for 46 drug ex vivo sensitivity on primary AML samples from diagnosis and relapse and found a "responder" group to many tyrosine kinase inhibitors. We examined the response to the multi tyrosine kinase inhibitor, dasatinib and the correlation with exome sequencing and transcriptome expression from our dataset and the Beat AML dataset. Mutations in FLT3-ITD and PTPN11 were enriched in the dasatinib sensitive samples as oppose to mutations in TP53 that were significantly more common in the resistance group to dasatinib (Fig 3). AML patients carrying PTPN11 mutations express higher level of LYN (a target of dasatinib) as compared to wild type. We found that LYN, HCK, CSK and EPHB2 (targets of dasatinib) genes were significantly overexpressed in the dasatinib responders carrying FLT3/ITD (Fig 4). Expression levels of these genes could predict which carriers of FLT3/ITD will respond to dasatinib . Finally, we demonstrated that dasatinib treatment targeted in vivo leukemic stem cells (LSCs) (Fig 5).
2. We are currently studying the mechanism which might lead to preleukemic deletions in myeloid malignancies. Results of this study will have an high impact on potentially aid in preventing somatic deletions that characterize clonal expansion in high risk individuals.