Dihydroorotate dehydrogenase inhibition acts synergistically with tyrosine kinase inhibitors to induce apoptosis of mantle cell lymphoma cells

Abstract Mantle cell lymphoma (MCL) is a non‐Hodgkin lymphoma that remains incurable with the treatment options available today. In the present study, we have identified the dihydroorotate dehydrogenase (DHODH), an essential enzyme for the de novo biosynthesis of pyrimidine‐based nucleotides, to be overexpressed in MCL in comparison to healthy peripheral blood mononuclear cells (PBMC). In vitro inhibition of the DHODH activity using a newly developed DHODH inhibitor, namely (R)‐HZ05, can induce MCL cell death in the nanomolar range independently than the P53 status of the investigated cell lines. Moreover, the combination of (R)‐HZ05 with tyrosine kinase inhibitor shows the synergistic activity on cell death. Pre‐clinical investigation on the efficacy of (R)‐HZ05 shows that it can be prolonged animal lifespan similar to ibrutinib. (R)‐HZ05 use in combination with tyrosine kinase inhibitor demonstrated a superior efficacy on tumor burden reduction and survival than either drug alone. We have demonstrated that the depletion of the pyrimidine nucleotide pool, using DHODH inhibitor, represents a new therapeutic strategy that may benefit MCL patients.


INTRODUCTION
Mantle cell lymphoma (MCL) is a non-Hodgkin lymphoma which remains incurable with the treatment options available today. Despite the good response rate to upfront chemotherapy, most patients relapse, presenting with an overall survival of 3-5 years [1][2][3][4]. The majority of MCL patients have the t(11;14)(q13;q32) translocation which leads to a deregulation of the cell cycle through the overexpression of cyclin D1 [5]. Along with oncogenic alterations in different cellular pathways (STAT3, NF-κB, AKT, Wnt, INK4a-CDK4-RB1) [2,3], the deregulation of the cell cycle leads to an aggressive and highly proliferative disease that demands a constant supply of nucleotides for RNA transcription and DNA replication [1]. Targeting metabolic pathways is one of the most successful strategies in anti-cancer therapies [6].
Recently, Ladds et al. characterized a novel dihydroorotate dehydrogenase (DHODH) inhibitor, namely (R)-HZ05, that increases the sensitivity to chemotherapy in acute myeloid leukemia (AML) cells [7]. DHODH is an essential enzyme for the de novo biosynthesis of pyrimidinebased nucleotides [6]. DHODH catalyses the conversion of dihydroorotate into orotate, which in turn is converted through several catalytic steps into deoxycytidine triphosphate, deoxythymidine triphosphate, and deoxyuridine triphosphate. Inhibition of DHODH has been linked to S-phase arrest and apoptosis induction through p53 upregulation [7] and his regarded as a new therapeutic target in cancers [8,9]. Importantly, as in AML, many MCL patients present with wild-type p53 [10,11]. Therefore, we hypothesize that targeting DHODH activity in MCL, using (R)-HZ05, as a single-agent and as a combinatory agent, represents a promising and yet unexplored strategy in MCL.  Figure 1B). Also, the protein expression of DHODH is higher in MCL cell lines ( Figure 1C) and MCL primary cells ( Figure 1D) when compared to PBMCs from healthy donors. Collectively, these observations suggest that DHODH is a potential target for inhibition in MCL.

RESULTS AND DISCUSSION
To NF-κB activation was not altered following (R)-HZ05 treatment ( Figure   S1C, D). We thus hypothesized that subsequent inhibition of DHODH and NF-κB signaling, an important signaling pathway for the proliferation and growth of MCL cells, might be of therapeutic value [12].
Since it has been suggested that inhibition of DHODH increases the chemosensitivity of cancer cell lines, we decided to investigate the effect of combining DHODH inhibition with a tyrosine kinase (TK) inhibitor that block NF-kB activation [6]. We tested ibrutinib, a Burton It has been reported that some TK inhibitors can reduce the intracellular uptake of uridine in a dose-dependent manner [13]. To evaluate if bemcentinib can prevent uridine uptake in cells, which may explain the synergism seen in combination with (R)-HZ05, we performed a uridine uptake experiment. The uridine uptake in the melanoma cell line ARN8 is greatly reduced in a concentration-dependent manner when treated with bemcentinib, suggesting that TK inhibition may prevent uridine transport also in MCL cells ( Figure 2D).

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. All authors contributed to the writing of the paper.

ETHICAL APPROVAL AND CONSENT TO PARTICIPATE
The use of human tissues and animal experiments have been approved by the Norwegian (REK number 2012/2245) and French institutional ethics committees.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.