Acute Myeloid Leukemia (AML) is caused by a variety of genetic aberrations; FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in AML at a frequency of 30%, determining a poor prognosis with high rates of relapse. FLT3 inhibitors used as monotherapy caused initial remission but ended in disappointing final results with relapses emerging in a short time. Studies aimed at elucidating the mechanism of resistance showed that it is due not only to additional mutations in FLT3 but also to development of clones with non-related mutations (for example involving the RAS pathway). Recent studies suggest that a possible strategy is to combine multiple approaches targeting different adaptive pathways, one of the most promising being to combine FLT3 inhibitors with inhibitors of the anti-apoptotic protein bcl-2. However, also in this case preliminary data suggest that FLT3-ITD+ AML cells are able to develop resistance.
We set up a therapeutic strategy based on the induction of ER and oxidative stress that shows cytotoxic activity against FLT3-ITD+ AML, either cell lines and primary blasts isolated from patients, mediated by generation of oxidative stress and by impairment of the adaptive response to ER stress. It relies on the combination of low doses of the differentiating agent retinoic acid (RA), the ER stress inducer tunicamycin (Tm) and the oxidative stress inducer arsenic trioxide (ATO) and importantly does not show toxicity on normal hematopoietic progenitors treated ex vivo. More recently, to facilitate possible translational applications, we substituted Tm with the proteasome inhibitor bortezomib (Btz) to induce ER stress and alteration of proteostasis, obtaining similar results. Indeed RA and ATO are employed in clinical practice for acute promyelocytic leukemia and Btz for multiple myeloma and mantle cell lymphoma. Here we propose to exploit the combination RBA to overcome FLT3-ITD AML resistance to FLT3 or bcl-2 inhibitors.
A key point in the Horizon2020 program ("Curing, treating disease: transferring fundamental knowledge to clinical practice") is aimed at improving the quality and duration of life for the European citizens. Fighting cancer is one of the top research priorities within the European Union. Progress in the treatment and outcomes for patients with acute myeloid leukaemia is challenging as this is a disease of predominantly older patients; many older adults, some of whom may exhibit chronic multi-organ dysfunction or poor performance status, cannot tolerate intensive chemotherapy and harbour leukemic cells that are inherently more resistant. AML is currently treated using the same drugs as 40 years ago, albeit with improved administration protocols. Since this disease is still lethal in the vast majority of cases, there is a clear need for novel therapies, especially in relapsing patients. Our project aims at improving inhibitors-based treatment of AML, in order to overcome resistance and to provide novel therapeutic options for AML.
Findings available in literature show that oxidative and proteotoxic stress achieve synergic effects with FLT3 and bcl-2 inhibitors (1-5). The advantage of adopting the combination RBA to generate such stresses reside in the fact that we observe high cytotoxicity already without FLT3 or bcl2 inhibitors, using low doses of each drug, that show no effects when the drugs are used alone. We are concluding the research project we focused on in the past three years, that aimed at examining the combination RBA as a therapeutic strategy for FLT3-ITD+ AML. In this context we are testing the combination RBA in vivo, in a "humanized" mouse model, in which MOLM13 cells are injected intravenously in the immunocompromised mouse NSG, to generate an orthotopic model of leukemia. As a control of toxicity of the combination, we treated wt mice for three weeks with the combination RBA. Histological analysis of organs isolated from these mice, in particular brain, liver, pancreas, spleen and kidney, demonstrated that the combination does not induce any sign of systemic toxicity. Furthermore these compounds are already administered in clinical practice at higher concentration than those we use. Obviously the situation could be different by combining RBA with FLT3i or venetoclax. However, as shown in Figure 2, FLT3i show a strong synergistic effect on MOLM13 cells, although these were not resistant clones, and these should allow decreasing the concentrations of the inhibitors as well. Altogether, our data and the knowledge available in literature suggest that RBA is an intriguing candidate to overcome resistance of FLT3-ITD+ AML to FLT3 inhibitors and bcl-2 inhibitors, provided either by rise of resistant clones that develops additional mutations along treatment or by the protection granted by the bone marrow mesenchymal cells.
References
1. Beeharry, N. et al. Blood Adv 3, 3661-3673 (2019).
2. Rodina, A. et al. Nature 538, 397-401 (2016).
3. Sallmyr, A. et al. Blood 111, 3173-3182 (2008).
4. Zong, H. et al. Cell Rep 13, 2159-2173 (2015).
5. Jayavelu, A.K. et al. Leukemia 30, 473-483 (2016).