The Hyperbolic NAMPT Inhibitor RPT1G Synergizes with BCL-2 Family Inhibitors and Helps Overcome Venetoclax Resistance in Acute Myeloid Leukemia Cells
Authors: Crimmins Gregory , Wu Min , Roushar Francis , Ramos Aguilar Claudia F. , Gharghabi Mehdi , Cole Banumathi , Lapalombella Rosa , De Jesús-Díaz Dennise A. 
Published: December 2024

Acute myeloid leukemia (AML) is the most common type of adult acute leukemia that progresses rapidly without treatment. Despite the emergence of new therapy options, most AML patients still face dire prognosis, with a 5-year survival rate of 37.5%. Venetoclax is a highly selective and effective B-cell lymphoma-2 (BCL-2) inhibitor that has demonstrated encouraging outcomes in AML patients, especially in those unfit for conventional chemotherapies. However, venetoclax-based regimens are not curative, and most patients will eventually develop resistance and relapse.

As the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, which converts nicotinamide (NAM) into NAD+, nicotinamide phosphoribosyltransferase (NAMPT) plays crucial roles in cancer metabolism. NAM metabolism mediates venetoclax resistance in relapsed leukemic stem cells (LSCs), making NAMPT an attractive target for overcoming such resistance. We have previously introduced RPT1G, a novel hyperbolic NAMPT inhibitor that achieves significant efficacy in acute lymphoblastic leukemia while avoiding severe on-target toxicities that limited clinical development of previous NAMPT inhibitors. Here we further explore the use of RPT1G in combination strategies that can aid in the efforts to treat venetoclax resistance in AML patients.

Cell health and apoptosis assessments revealed significant synergy between RPT1G and venetoclax in AML cells that are sensitive to venetoclax treatment, such as MV4;11, Molm 13, and ML-2, as well as venetoclax-resistant cells, including OCI-AML3 and THP1. Notably, synergy with venetoclax was also observed in cells that are resistant to RPT1G monotherapy, including HL-60 and Kasumi-1 cells. Moreover, we developed a venetoclax-resistant MV4;11 cell line (MV4;11-VR) showing over 50-fold increase in IC50 to venetoclax and observed strong synergy between RPT1G and venetoclax in these cells. Further analyses revealed that RPT1G and venetoclax synergized to reduce total cellular NAD+ in MV4;11 cells. While RPT1G exhibits mostly additive or modestly synergistic effects with AML standard-of-care agents, cytarabine and azacitidine, the synergy between RPT1G and venetoclax was maintained in MV4;11 cells in the presence of either agent. Moreover, RPT1G strongly synergizes with olaparib, a poly(ADP-ribose) polymerase inhibitor, and olaparib further enhanced the synergy between RPT1G and venetoclax.

In contrast, RPT1G and venetoclax showed mostly additive or antagonistic effects in cultured human peripheral mononuclear cells from healthy donors. As RPT1G exhibits a favorable safety profile in animal toxicology studies and none of the adverse hematological toxicities of venetoclax, the use of this combination strategy could lower the dose of venetoclax required to achieve efficacy against AML cells and increase the safety margin of venetoclax-based regimens.

B-cell lymphoma-extra large (BCL-XL) and myeloid cell leukemia 1 (MCL-1) are targets of interest to overcome venetoclax resistance, however, there are concerns over elevated on-target toxicities. We observed significant synergies between RPT1G and navitoclax, an inhibitor to multiple BCL-2 family proteins, as well as RPT1G and the MCL-1 inhibitor AMG-176. Of note, navitoclax or AMG-176 further enhanced the synergy between RPT1G and venetoclax in MV4;11-VR cells.

The efficacy of the RPT1G and venetoclax combination was further evaluated in a MV4;11-luc mouse xenograft model. At doses of RPT1G and venetoclax that exhibited modest effects as monotherapies, the combination of the two agents significantly reduced the leukemia burden in mice, while showing no observable toxicities.

In summary, the first-in-class hyperbolic NAMPT inhibitor RPT1G exhibited strong synergy with agents targeting anti-apoptotic BCL-2 family proteins in AML cells, both alone and in the presence of other standard-of-care agents. Given the crucial roles of NAMPT and BCL-2 family proteins in the survival and treatment-resistance of LSCs, RPT1G is emerging as an ideal candidate to be included in novel combinatorial therapies to enhance treatment efficacy of BCL-2 family inhibitors and overcome therapy resistance, improving overall outcome in a wide range of patients with AML and potentially other hematological malignancies.