Pharmacological Modulation of SAMHD1 Activity by CDK4/6 Inhibitors Improves Anticancer Therapy

Abstract

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a dNTP triphosphohydrolase involved in the regulation of the intracellular dNTP pool, linked to viral restriction, cancer development and autoimmune disorders. SAMHD1 function is regulated by phosphorylation through a mechanism controlled by cyclin-dependent kinases and tightly linked to cell cycle progression. Recently, SAMHD1 has been shown to decrease the efficacy of nucleotide analogs used as chemotherapeutic drugs. Here, we demonstrate that SAMHD1 can enhance or decrease the efficacy of various classes of anticancer drug, including nucleotide analogues, but also anti-folate drugs and CDK inhibitors. Importantly, we show that selective CDK4/6 inhibitors are pharmacological activators of SAMHD1 that act by inhibiting its inactivation by phosphorylation. Combinations of a CDK4/6 inhibitor with nucleoside or folate antimetabolites potently enhanced drug efficacy, resulting in highly synergic drug combinations (CI < 0.04). Mechanistic analyses reveal that cell cycle-controlled modulation of SAMHD1 function is the central process explaining changes in anticancer drug efficacy, therefore providing functional proof of the potential of CDK4/6 inhibitors as a new class of adjuvants to boost chemotherapeutic regimens. The evaluation of SAMHD1 expression in cancer tissues allowed for the identification of cancer types that would benefit from the pharmacological modulation of SAMHD1 function. In conclusion, these results indicate that the modulation of SAMHD1 function may represent a promising strategy for the improvement of current antimetabolite-based treatments.

Keywords: CDK4/6; HIV; SAMHD1; antimetabolite; cancer; pemetrexed.

Figure 1

(A) Degradation of sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) by HIV-2 Vpx enhances HIV-1 replication in monocyte-derived macrophages (MDMs). MDMs previously treated or not with HIV-2 Vpx were infected with a VSV-pseudotyped HIV-1 GFP virus and replication assessed 2 days later by measuring GFP expression. A 5-fold change in HIV-1 replication was observed after Vpx-mediated SAMHD1 degradation. Mean ±SD of ten independent donors performed in duplicate is shown. A representative western blot showing degradation of SAMHD1 expression in MDMs after Vpx treatment is also shown. (B) Decreased sensitivity of AZT after Vpx-mediated SAMHD1 degradation in MDMs. Dose response of the NRTI AZT and NNRTIs NVP, in wild-type (■) or SAMHD1-depleted (Δ) MDMs. Inhibition of HIV infection was measured as the percentage of GFP+ cells relative to the no drug condition. Mean ±SD of at least ten independent donors performed in duplicate is shown. (C) SAMHD1 modifies antiviral activity of AraC. Dose response of the AraC in wild-type (■) or SAMHD1-depleted (Δ) MDM. Inhibition of HIV infection was measured as the percentage of GFP+ cells relative to the no drug condition. Mean ±SD of at least three independent donors performed in duplicate is shown. Mean ±SD of at least three independent experiments performed in triplicate is shown. * p < 0.05; ** p < 0.005; *** p < 0.0005.

Figure 2

SAMHD1 modifies antiviral and activity of antimetabolites. Dose response of the nucleoside analogues (A) or anti-folate drugs (B) currently used as anti-cancer treatments in wild-type (■) or SAMHD1-depleted (Δ) MDMs. Inhibition of HIV infection was measured as the percentage of GFP+ cells relative to the no drug condition. Mean ±SD of at least three independent donors performed in duplicate is shown. * p < 0.05; ** p < 0.005; *** p < 0.0005.

Figure 3

Antiviral efficacy of CDK4/6 inhibitors depends on SAMHD1 expression. (A) Dose response of the CDK4/6 inhibitor Palbociclib, in wild-type (■) or SAMHD1-depleted (Δ) MDM. Inhibition of HIV infection was measured as the percentage of GFP+ cells relative to the no drug condition. Mean ±SD of at least ten independent donors performed in duplicate is shown. (B) CDK4/6 inhibitors lose antiviral activity in SAMHD1-depleted macrophages. As in (A), dose response of two other CDK4/6 inhibitors, ribociclib (left panel) and abemaciclib (right panel), in wild-type (■) or SAMHD1-depleted (Δ) MDMs. Mean ±SD of two independent donors performed in duplicate is shown. (C) Palbociclib blocks SAMHD1 inactivation by phosphorylation. Western blot analysis of lysates of untreated MDMs (no drug, ND) or macrophages treated with palbociclib at the indicated doses. Membranes were blotted with an anti phospho-SAMHD1 antibody, total SAMHD1, anti phosho-pRB and total pRB. Hsp90 antibody was used as control. A representative donor is shown. * p < 0.05; ** p < 0.005; *** p < 0.0005.

Figure 4

Pharmacological activation of SAMHD1 enhances antiviral activity of antimetabolites. (A) Relative effect of the combination of palbociclib-pemetrexed measured as antiviral activity. Inhibition of HIV infection with increasing doses of palbociclib and pemetrexed was measured. Percentage of GFP+ cells relative to the no drug condition is shown in presence (left panel) or absence (right panel) of SAMHD1. Mean ±SD of at least three independent donors performed in duplicate is shown. (B) As in (A), relative effect of pemetrexed alone (white bars) or in combination with a fixed dose of palbociclib 0.04 µM (black bars), in the presence (left panel) or absence (right panel) of SAMHD1. Mean ±SD of at least three independent donors performed in duplicate is shown. (C) Relative effect of the combination of midostaurin-pemetrexed measured as antiviral activity. Inhibition of HIV infection with increasing doses of midostaurin and pemetrexed was measured. Percentage of GFP+ cells relative to the no drug condition is shown in presence (left panel) or absence (right panel) of SAMHD1. Mean ±SD of at least three independent donors performed in duplicate is shown. (D) As in (C), relative effect of pemetrexed (PTX) alone (white bars) or in combination with a fixed dose of midostaurin 0.2 µM (black bars), in the presence (left panel) or absence (right panel) of SAMHD1. Mean ±SD of at least three independent donors performed in duplicate is shown. PD, palbociclib; PTX, pemetrexed; MID, midostaurin. * p < 0.05; ** p < 0.005; *** p < 0.0005.

Figure 5

Pharmacological activation of SAMHD1 enhances cytotoxicity of antimetabolites. (A–C) Effect on cell viability of palbociclib–pemetrexed combination in TZM-bl, T47D and MDA-MB-468 cell lines, respectively. Left panels, cytotoxic activity of palbociclib alone (5 µM, white bars), pemetrexed alone (black bars at 0.2, 1 or 0.04 µM for TZM-bl, T47D and MDA-MB-468 respectively) or the combination of both drugs at the same concentration (grey bars). Right panels, cytotoxic activity of palbociclib alone (5 µM, white bars), fluorouracil alone (5 µM, black bars) or the combination of both drugs at the same concentration (grey bars). Drug concentrations were chosen depending calculated CC₅₀ under specific experimental conditions (Table S1). * p < 0.05; ** p < 0.005; *** p < 0.0005. PD, palbociclib; PTX, pemetrexed; FU, 5-fluorouracil.

Figure 6

Regulation of dNTP pool is responsible for drug synergy. (A) Protein expression in MDMs treated with palbociclib (PD) at 1 µM, pemetrexed (PTX) and fluorouracil (FU), both at 5µM and the corresponding drug combinations PD+PTX and PD+FU. Hsp90 was used as a loading control. A representative blot is shown. (B) Proposed model of drug interactions. Antimetabolites affecting dNTP synthesis such as pemetrexed inhibit dNTP pool by a mechanism not directly affecting SAMHD1 activation and thus synergy with anticancer drugs affecting SAMHD1 phosphorylation as palbociclib is higher compared to compounds also targeting SAMHD1 function (i.e., fluorouracil) or exclusively affecting SAMHD1 (i.e., midostaurin). As a consequence, the antiviral and cytotoxic efficacy of antimetabolites is significantly enhanced when used in combination in vitro.

Figure 7

Expression of SAMHD1 protein in tumor samples from cancer patients. (A,B) IHC staining of SAMHD1 in pancreas (A) and lung (B) tumor samples. Morphology of tumor cells is shown by routine hematoxylin stain of paraffin embedded tumor sections. SAMHD1 is stained in brown. Original magnification ×200. Scale bar, 20 µm. (C) Percentage of tumors expressing SAMHD1, depending on its relative expression in IHC as classified in Human Proteome Atlas (www.proteinatlas.org, IHC data of SAMHD1 expression from 17 different tumor types were retrieved and classified according to protein expression levels. ND, not detected.