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Objectives

Thousands of adults are diagnosed with acute myeloid leukemia (AML) in Europe annually, with a median age of 67 years. Despite advances in therapeutics and supportive care, the majority of patients with AML die from their disease. For most subtypes of AML, the mainstay of initial treatment was developed nearly 40 years ago as a combination of cytosine arabinoside (ara-C) with an anthracycline, and this regimen remains the worldwide standard of care. Approximately 70%-80% of patients achieve complete remission, but most ultimately relapse. Thus, the overall survival is only 40%-45% 5 years after the initial diagnosis. Among patients, almost half of those with a good performance status can achieve complete remission, but cure rates are 10% and the median survival is just 1 year. The outlook for older AML patients has not changed in 3 decades, and is even worse for older patients with unfavorable cytogenetics and poor performance status.

Therefore, it would be helpful to improve the diagnostic and treatment accuracy by developing novel markers or drugs that could improve the therapeutic ratio and clinical outcome. First described in C. elegans more than a decade ago, over 21,000 members of a new class of small ncRNAs, named microRNAs (miRNAs), have been identified in the last decade in vertebrates, flies, worms and plants, and even in viruses. In humans, the miRNoma (defined as the full spectrum of miRNAs) contains more than 1500 experimentally cloned miRNAs and the total number is expected to overpass the two thousands mark. It has been speculated that miRNAs could regulate ~30% of the human genome. The list of proposed miRNA functions includes hematopoietic B-cell lineage fate (miR-181), B-cell survival (miR-15a and miR-16-1) or cell proliferation control (miR-125b and let-7). The small difference between incidence and mortality rates is a clear mirror of the fact that AML is still a lethal disease. Advanced disease at presentation and the intrinsic agressiveness are the main causes of the increased mortality rate. Therefore, it would be helpful to improve the diagnostic and prognostic accuracy by identifying readily accessible molecular markers that predict some of the variation in clinical outcomes. MicroRNA (miRNA) alterations are involved in the initiation and progression of any type of human cancer, including AML. MicroRNA expression profiling of human tumors has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNAs genes that may represent downstream targets of activated oncogenic pathways or that are targeting protein coding genes involved in cancer. Binding various anti-FLT3 drugs to nano-carriers offers a new and exciting approach to an early diagnosis for acute leukemia. When diagnosis is achieved, chemotherapy often fails and the clinician only has the alternative of palliation. But a more precise, targeted delivery of anti-cancer drugs may lead to an improved therapeutic ratio. Such a tergeted delivery may be possible by functionalizing gold nanoparticle (GNP) with various drugs already used in the clinic, as malignant cells differentially internalize GNPs in comparison with healthy cells, as already proven by our research team in the past three years. This leads to a higher drug concentration in the cancer cell, with lower systemic sideeffects. We chose to focus on miRs that are downregulated in AML. Their downregulation in cancer suggests that they may play a tumor suppressive role. The fact that normal tissues already express these miR species suggests that further upregulation – in case of off-target effects- may not have a detrimental result.

We have already performed miR microarrays on 16 AML and 16 myelodysplastic syndrome cases (MS). The raw data were 75% normalized and miRs with expression level of less than 50 microarray raw units in MS were not included in the analysis. A total number of 77 miR species demonstrated significant expression in MS specimens.

MiRs displaying at least a 2-fold downregulation in AML compared to MS were chosen for further analyses. A total number of 7 miR species fulfilled these criteria. We selected miR-375 and miR-638 for further analyses, based on the fold downregulation in AML vs. MS, as well as based on a higher level of expression in MS. The microarray results were then verified for miR-638. We performed real time RT-PCR on 10 specimens of MS and 10 specimens of AML. As shown by our previous studies, miR-638 is significantly downregulated in AML. The downregulation of miR-638 in AML suggests that this miR may have a tumor suppressive role. To explore this hypothesis, we transfected AML cells OCI-AML3 with miR-638 and with the control Non-Specific Mimic (NSM). Cells treated with miR-638 grew slower than cells treated with control, indicating a possible role for miR-638 in suppressive AML growth.

In a proof of principle study, before we started experiments with miR-638, we also performed cell proliferation and cell cycle experiments with miR-494 in the same leukemia cell line (OKI-AML3). Similar to miR-638, miR-494 is downregulated in human AML and its transfection in cell lines decreases the growth of cancer cells. We aimed at quantifying the effects of miR-494 on proliferation. Therefore, we performed BrdU incorporation experiments in cells treated with miR-494 or with NSM. We found a statistically significant decrease in AML cell proliferation as a result of miR-494 treatment. To further dissect the mechanisms of decreased proliferation following treatment with miR-494, we performed cell cycle analyses. We found that miR-494 induces statistically significant cell cycle arrest in G1, suggestive of a reinforcement of the G1-S checkpoint (Figure 3). After identification of the target miRs, the projects aims at validating a new prognostic panel in comparison with FLT3 mutation, a standard-of-care in AML prognosis. The validation will be carried out on a number of 30 cases of AML patients at the Ion Chiricuta Institute of Oncology. The young physicians and clinicians will have the opportunity to learn the basics of molecular hematology, with great impact in the development of their continuous medical education. A large number of AML cases will relapse and clinicians currently use the FLT3 inhibitors lestaurtinib, midostaurin, sorafenib and quizartinib for therapy. Our previous publications support the concept of targeted delivery of drugs using nanovectors. We plan to functionalize these drugs with nano-carriers and asses the effects of the newly described structures both in vitro and in vivo. This step will be carried out in collaboration with the Babes Bolyai University and the University of Bergen, aiming at training young researcher from these institutions with the latest protocols and research techniques in molecular oncology and nanotechnology. The Norwegian partner will also provide training for young investigators and PhD students from Romania in developing new experimental designs based on animal models of AML, thus establishing the basics of a strong collaboration between Norway and Romania.