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Dissecting to hit the therapeutic targets in nucleophosmin (NPM1)-mutated acute myeloid leukemia

Periodic Reporting for period 4 - ContraNPM1AML (Dissecting to hit the therapeutic targets in nucleophosmin (NPM1)-mutated acute myeloid leukemia)

Reporting period: 2021-10-01 to 2022-03-31

Acute myeloid leukemias (AML) is an aggressive hematological malignancy most commonly affecting elderly population and an unmet medical need. AML with nucleophosmin (NPM1) gene mutations is a new well recognized entity in the World Organization of Health (WHO) classification of myeloid neoplasms and accounts for about one-third of AML in adult patients. A therapy specific for this frequent leukemia has not been yet identified.

‘ContraNPM1AML’ project aimed to dissect and hit therapeutic targets in NPM1-mutated AML – hence, the acronym of the project. The key challenging issues were how to unravel its unique vulnerabilities and tailor a therapy. ContraNPM1AML strategy was based on two complementary approaches, each focused on either the target or the drug. The hypothesis-driven approach pointed mainly to re-think, re-explore the ‘known’ in the light of the specific genetic lesion (i.e. NPM1 mutation), and was based on previous and preliminary observations done by our research group in the past years. In parallel, a wide screening-based approach with application of novel technologies and more comprehensive analyses – aimed to the ‘unknown’ – was adopted to identify new therapeutic targets specific for the disease. This was the part of the project certainly at higher risk, but required by the complexity of NPM1 as multitasking protein and of the newly established intracellular pathways in the leukemic cells as a consequence of its mutation. The end goal of this research project was to better understand what sustains leukemia in NPM1-mutated AML subtype, and to identify its Achilles’ heel and its counterpart, specifically drugs that only kill or kill more selectively AML cells with NPM1 gene mutations.

ContraNPM1AML is now concluded. The outputs of the project confirm the success of the designed strategy. The contribution of ContraNPM1AML will go well beyond the time frame of the project. Indeed its results open new research horizons and new avenues for designing novel tailored therapies and clinical trials with final direct benefit to patients.
ContraNPM1AML provides evidence on the role of specific genes involved in leukemia and on the peculiar activity against NPM1-mutated AML of a series of drugs, approved for other diseases, that therefore have been ‘repurposed’, within the project, in the setting of NPM1-mutated AML. Here we confirm specific actions of arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA) - two drugs used for the treatment of another form of leukemia, named promyelocytic leukemia – in NPM1-mutated AML, where they induce degradation of NPM1 oncoprotein and downregulation of HOX genes, a pathway critical in leukemia renewal, supporting the concept of the essentiality of NPM1 mutant in the leukemia maintenance through HOX genes regulation (Brunetti et al, Cancer Cell 2018). On HOX genes and NPM1 oncoprotein interplay, using the drugs FLT3-inhibitors in FLT3-ITD and NPM1-mutated AML patients we showed for the first time terminal cell differentiation of leukemic cells ‘uncoupled’ from the HOX-related ‘self-renewal signature’ that remained ‘activated’ (up) in presence of sustained NPM1 oncoprotein expression, supporting again its key role in HOX maintenance. These novel data have not been yet published, but were presented at either national or international scientific meetings. The studies on the nucleolar stress inductor drug dactinomycin, that we have previously shown to be clinically active in patients with NPM1-mutated AML, were concluded and showed that expression of NPM1 mutant lowers the threshold for stress-induced cell death, providing an explanation for its clinical activity in NPM1-mutated AML (Gionfriddo et al, Leukemia 2021). Moreover, in collaboration studies, we unveiled a new interplay between NPM1 oncoprotein, the tumour suppressor PML and mitochondria in mediating dactinomycin response and synergistic effect with the bcl2-inhibitor venetoclax (Wu et al, Cancer Discov 2021). Based on our results, dactinomycin is deserving further investigation in larger clinical studies and in combinatorial settings. We also studied the role of omacetaxine mepesuccinate, a drug that inhibits protein synthesis, with the purpose of targeting levels of proteins relevant for leukemia cell survival and, strikingly, we found that it decreased NPM1 oncoprotein levels, besides other relevant players in leukemia, and induced leukemia cell differentiation. Its combination with the bcl-2 inhibitor venetoclax showed unexpected potent and synergic anti-leukemic effects in preclinical studies so that these findings were readly translated from bench to bedside in the first clinical trial stemmed from our ContraNPM1AML ERC-granted research and based on the chemo-free association of these two drugs.

Finally, a substantial effort was placed in two main tasks to discover new targets by identifying essential interactions by high throughput drug library screens and gene targeting in NPM1-mutated AML. To carry out the highly complex drug screening the ERC team was supported by the Horizon 2020-funded CORBEL project, that guaranteed connection with two European research infrastructures, EU-OPENSCREEN (FMP, Berlin) and Euro-BioImaging (EBML, Heidelberg), allowing the access to technologies not available in the PI’s lab and to scale-up screening to a 40.000 compounds library. Despite the COVID-19 pandemic, a lockdown, and constantly-evolving travel restrictions have slowed down cross-border cooperation, the ERC/Corbel team succeeded in completing the chemical screening, leading to the identification of two main classes of drugs that have been chosen for the further investigational studies within the time frame of the project and were proven to be particularly effective in NPM1-mutated AML in the preclinical setting. The screening lead also to the identification of other drugs and even not-annotated compounds that will be the object of future drug development studies.
Among the most relevant results beyond the state of the art is the observation that specific inhibitors of FLT3 drive in NPM1-mutated AML a clonal multilineage terminal cell differentiation of the leukemic cells that is uncoupled from HOX gene expression and mimics complete response. This finding has biological relevance in understanding pathways supported by the pathologic FLT3 signal and could have a tremendous clinical impact since it could lead to a completely different way of defining and evaluating response to target therapy as well as designing study protocols including FLT3 and possibly other novel specific inhibitors in AML.
Another ground-breaking result is the discovery of a potent anti-leukemic activity and the underlying mechanisms of a novel chemo-free drug combination including venetoclax and omacetaxine mepessucinate in NPM1-mutated AML, that we have validated in preclinical models and translated into a clinical trial that is now recruiting patients.
Also the results of the drug screening campaign are ground-breaking since different classes of drugs have been identified as drugs with a more specific action in NPM1-mutated AML, opening new research and therapeutic avenues for this frequent leukemia. 

This was possible thanks to the virtuous interaction and strong motivation of the team which, although divided into subgroups dedicated to the different tasks of the project, worked in synergy beyond the limits set by the COVID19 pandemic.
Targeting dependencies in NPM1-mutated AML
Strategy of ContraNPM1AML