PIM447, a Pan-PIM Kinase Inhibitor, in Combination with Venetoclax Exerts a Potent Anti-Tumor Activity in the Bone Marrow Niche

Acute myeloid leukemia (AML), the most common adult leukemia, is characterized by the uncontrolled growth of abnormal myeloid cells. While AML therapy has seen progress over the last decade, relapse continues to be the most therapeutic challenge. To overcome this hurdle, novel targeted therapies based on dysregulated molecular biomarkers and pathways are urgently needed. PIM kinases, a family of proteins constitutively overexpressed in hematologic malignancies, have emerged as promising therapeutic targets. PIM kinases regulate cell proliferation and survival and inhibit apoptosis by activating anti-apoptotic pathways, such as BCL2, conferring a worse prognosis in AML patients and contributing to disease relapse. Therefore, specifically targeting PIM kinases holds significant potential in improving AML treatment outcomes. Here, we investigated the effects of a novel Pan-PIM kinase inhibitor, PIM447 (Novartis), in AML patient samples. We further explored the dual combination of PIM447 and Venetoclax, a BCL2-targeting chemotherapy drug, to evaluate their potential synergistic effect on AML treatment. To analyze the functional role of PIM447 in AML, bone marrow mononuclear cells (BMNCs) isolated from AML patients and healthy donors (HD) (n=5) and two human AML cell lines, Molm-13 (FLT3-ITD-mutated) and OCI-AML3 (NPM1-mutated) were treated with PIM447. PIM447 treatment selectively increased apoptosis rates (Annexin V⁺⁾ in AML BMNCs (57% decrease, p<.05) and human AML cell lines (91% decrease, p<.0001) without affecting HD cell death. The efficacy of PIM447 was tested in an organotypic time-lapse microscopy assay, using AML BMNCs cocultured with mesenchymal stromal cells (MSCs) in AIMV-10% FBS medium and 1.5 mg/ml of collagen I. PIM447 successfully inhibited AML cell viability in a dose-dependent manner. To validate this potent antineoplastic activity of PIM447 in vivo, leukemic NSG mice bearing Molm-13-GFP AML cells received daily intraperitoneal injections of the PIM447 (2mg/kg) for 5 days. PIM447 treatment diminished AML cells in the BM, peripheral blood, and spleen (60% decrease, all p<.05). To explore the applicability of PIM447 for combinatory therapy, AML cells (Molm-13 and OCI-AML3) were treated with PIM447 alone or in combination with the BCL2 inhibitor Venetoclax. Notably, PIM447 enhanced the cell death induced by Venetoclax (~90% increase, p<.0001) and diminished the levels of the anti-apoptotic BCL2, BCLXL, and MCL1 proteins (60% decrease, p<.0001). Furthermore, synergy analysis was performed using the Synergy Finder package in R to confirm the pharmacological interaction of this combinatory therapy (PIM447 and Venetoclax) for AML treatment. The results showed that the combination yielded an additive interaction between both compounds (Bliss, ZIP, Loewe, HAS, all synergy scores <10 and >-10). To understand the role of this combinatory treatment strategy within the BM perivascular niche, we established a 3D niche-mimicking model, where AML cells (Molm-13 or primary BMNCs from AML patients) were cocultured with MSCs and human umbilical vein endothelial cells (HUVECs) in a hemostatic sponge used as an extracellular matrix. The PIM447 treatment successfully counteracted the protective effects of these niche cells (endothelial cells and MSCs), by inhibiting the proliferation/growth of Molm-13 (70% decrease, p>.0001) and blasts from AML patients (17% decrease, p>.05). When combined with Venetoclax, the results showed a stronger anti-proliferative effect (Molm13: 90% decrease, p<0001; blasts: 60% decrease). Overall, our results demonstrated that PIM kinase inhibition (through the PIM447 compound) is a valuable tool for targeted AML therapy, exhibiting potent cytotoxic activity against AML cells. Notably, when combined with a BCL2 inhibitor, this strategy effectively overcomes the protective effect of the bone marrow microenvironment.

Funding: FAPESP 2017/21801-2, 2019/25247-5, 2021/05320-0; CNPq 303405/2018-0.