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

Periodic Reporting for period 2 - ContraNPM1AML (Dissecting to hit the therapeutic targets in nucleophosmin (NPM1)-mutated acute myeloid leukemia)

Reporting period: 2018-10-01 to 2020-03-31

Acute myeloid leukemias (AML) is an aggressive hematological malignancy most commonly affecting elderly population (median age reached 70 years). Incidence of the disease will rapidly rise due to the proportional increase of the aging population. AML is not a single disease but include a heterogeneous group traditionally difficult to classify and treat. The “one-size-fits-all” standard treatment with intensive chemotherapy allows in younger patients cure rates of about 40-50%. However, outcome of older patients remained very poor. Genomic and molecular characterization uncovered the widespread heterogeneity in AML and greatly added to our understanding of the disease biology. This led also to identification of new therapeutic targets and development and approval of new drugs targeting specific AML-associated genetic lesions that hold great promise.

Today NPM1-mutated AML is a new well recognized entity in the World Organization of Health (WHO) classification of myeloid neoplasms. However, this leukemia remains a medical need, since many patients, particularly older ones, succumb to their disease, and a therapy has not been yet identified.

‘ContraNPM1AML’ project focuses on NPM1-mutated AML and relies on the concept that to find a cure for AML we need to concentrate the view and efforts into a specific genetic subtype with a unique genetic lesion in order to uncover its specific dependences and find a specific therapy. This is the ambitious goal of our ERC-funded project.

The strategy includes either hypothesis-driven or screening-based approaches to either hit directly NPM1 mutant or identify disease-specific vulnerabilities (so called, ‘synthetic lethal’ interactions with NPM1 mutation) in order to: i) clarify and better understand mechanisms of development and/or maintenance of leukemia; ii) identify/exploit sensitive pathways and chemical compounds/drugs that kill specifically AML cells harbouring NPM1 mutations; iii) design and develop clinical trials to prove the safety and efficacy of promising new treatments and make them readily available to patients.

The findings of this project will address an urgent medical need that is to find a ‘tailored’ targeted therapy in NPM1-mutated AML.
On the side of the hypothesis-driven approach, taking advantages of our knowledge on the biology of the disease and previous data, and of specific cellular models of NPM1-mutated leukemia available and characterized in our laboratories, we tested the antileukemic effects and explored specific mechanisms of action of different drugs commonly used for other either hematological or non-hematological malignancies, with the purpose of drug repositioning. In particular, we found that arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA) - two drugs used for the treatment of a particular form of acute myeloid leukemia, named promyelocytic leukemia - are able to induce partial degradation of the NPM1 mutant leukemic protein, associated with the reversal of some phenotypic aspects linked to the NPM1 mutation itself. In particular, downregulation of the HOX genes, a molecular pathway critical in leukemia renewal and activated in NPM1-mutated AML, was relevant in explaining the final effect of growth inhibition and cell death. These data gave contribution to other experimental evidences, recently published, in supporting the concept of the essentiality of NPM1 mutant in the leukemia maintenance through HOX genes regulation. We are currently studying these drugs in combination with others, including actinomycin D that we have previously shown to be clinically active in patients with NPM1-mutated AML. With a similar approach and in the same models, we also identified the combination of omacetaxine and venetoclax, as extremely active in preclinical studies (data not yet published), and succeeded in attracting other funds in order to carry out the first clinical trial stemmed from our ContraNPM1AML ERC-granted research and based on the chemo-free association of these two drugs.
We have also exploited target therapy in patients as tool to answer biological and clinical questions. We analyzed samples from patients treated with specific inhibitors of FLT3, a pathway activated in many AML and involved in leukemia development, and found that they induce a terminal cell differentiation of the NPM1-mutated leukemic cells into ‘normal looking’ cells belonging to the different bone marrow lineages. From the biological point of view, this phenomenon highlights that FLT3 signal acts in NPM1-mutated AML in mediating a block of differentiation. From the clinical point of view, this differentiation, mimicking complete response at evaluation by standard approaches, leads to misinterpretation of the real patient status, thus integration with non-conventional methodologies is required to defining and evaluating response to target therapy. These data have not been yet published, but were presented at either national or international scientific meetings.

However, given the complexity of the intracellular pathways interactions in leukemic cells to increase the probability of the identification of new therapeutic targets specific for NPM1-mutated AML, we pursued a wide screening-based approach. Whilst with the planned genome-wide CRISPR knock-out screening, we had some scientific and technical issues so that so far we have worked mainly on re-defining our strategy and setting experimental conditions, major progresses have been made instead with the high-throughput screening (HTS) of drugs. Indeed, in collaboration with the Leibniz-Forschungsinstitut für Molekulare Pharmakologie we have performed the HTS of 38720 chemical compounds, including a set of drugs in clinical use, and identified 37 compounds/drugs selectively active on the NPM1-mutated leukemia cells, some of which belonging to the same class of drugs thus enforcing the results. Some of these drugs entered already the validation phase to have confirmation of their selective effect in a larger set of fit-on-purpose models in our laboratories, and the preclinical phase.
One of the most relevant results beyond the state of the art is the observation (manuscript submitted) that specific inhibitors of FLT3 drive in NPM1-mutated AML a clonal multilineage terminal cell differentiation of the leukemic cells that 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. We have already validated this combination in preclinical models (manuscript in preparation) and now translated it into clinic with the development of a clinical protocol for patients with relapsed/refractory disease, waiting for the final approval by the competent authorities in Italy. I expect to identify essential pathways in NPM1-mutated AML development and novel therapeutic targets in order to develop new drugs, and to identify other therapeutic combinations and develop new clinical trials for the benefit of patients.