ATRAID regulates the action of nitrogen-containing bisphosphonates on bone

Abstract

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFFs) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase (FDPS), resulting in defects in protein prenylation. Yet it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID. Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Lastly, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare non-synonymous coding variants in patients with ONJ or AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

One Sentence Summary:

ATRAID is essential for responses to the commonly prescribed osteoporosis drugs nitrogen-containing bisphosphonates.

INTRODUCTION

Nitrogen-containing bisphosphonates (N-BPs) are the standard treatment for osteoporosis and several other bone diseases (1, 2). Certain N-BPs (pamidronate, zoledronate) are also routinely prescribed to prevent skeletal complications in patients with multiple myeloma and with bone metastases from other malignancies, including breast and prostate cancer (3). However, because N-BPs cause rare yet serious side-effects, such as atypical fractures (AFFs) and osteonecrosis of the jaw (ONJ), many patients avoid taking them (1, 4–6), causing the number of prescriptions to plummet over 50% in the last decade (6, 7). A plan for addressing this crisis, developed by American Society for Bone and Mineral Research (ASBMR) leadership, calls for better pharmacogenomics to identify genetic factors that may underlie response to this class of drugs (6).

A goal of personalized medicine is to identify biomarkers that underlie drug responsiveness. For N-BPs, it can be said that there are limited personalization options owing to the limited number of genes implicated in the pharmacologic effects of N-BPs. Exposure of cells to N-BPs leads to inhibition of farnesyl diphosphate synthase (FDPS, also known as FPPS) resulting in reduction in protein prenylation (8). On the basis of this observation, it is widely believed that N-BPs act therapeutically by impairing protein prenylation, ultimately leading to deficits in numerous cellular processes including differentiation, recruitment, and adhesion of osteoclasts (the major bone resorptive cell type) to bone and/or osteoclast cell death (9–11).

Recently we performed CRISPRi-based, genome-wide screening and identified a poorly characterized gene, SLC37A3, that provides molecular details for how N-BPs reach their target, FDPS (12). As part of that work we determined that SLC37A3 requires another poorly characterized protein, ATRAID, for its expression (12). Here, we independently identified ATRAID using a different genome-wide, mutagenesis strategy. We generated Atraid-deficient mice and determined that it is required for the regulation of N-BPs on bone. We also performed exome sequencing in patients taking N-BPs and identified and functionally validated rare coding variants in ATRAID in patients that suffered side effects, namely atypical femoral fractures and osteonecrosis of the jaw.

RESULTS

ATRAID is required for molecular responses to nitrogen-containing bisphosphonates

To provide insight into the mechanism(s) of N-BPs action, we performed a genetic screen to identify human genes required for the anti-proliferative effects of N-BPs (Fig. 1A). We used a largely haploid human cell line of myeloid origin (KBM7, also known as HAP1) to generate a library of retroviral gene trap mutants (13) and then selected for clones that are resistant to cytotoxic concentrations of alendronate (ALN). The advantages of this cell line for genetic screening include: i) each gene is present as a single copy, enabling gene inactivation (except those genes on chromosome 8); ii) KBM7 cells are human and of the hematopoietic lineage, increasing the likelihood that any genes we identify could be relevant to the natural context for N-BPs, the bones of human patients (14); and iii) it is a different cell line and a different mutagenic approach than used previously with CRISPRi in K562 cells (12), which allows us to independently assess those results. Using this haploid approach, we identified, ATRAID, also known as APR-3/C2orf28 (15), as the gene most significantly enriched for insertions in alendronate-resistant cells compared to untreated cells (FDR corrected P-value = 7.02e-45) (Fig. 1B; fig. S1, A and B, and table S1). Providing confidence in our screen, we also identified SLC37A3, as well as SNTG1, PLCL1, and EPHB1, which have been previously connected to N-BP action on bone cells and/or human bone diseases (table S1) (16–18).

Fig. 1. ATRAID is required for molecular responses to nitrogen-containing bisphosphonates.

(A) Schematic of haploid mutagenesis screening pipeline. Sequencing-based identification of gene-trap insertion sites in alendronate-resistant human haploid KBM7 cells. Genomic DNA for sequencing was obtained from mutagenized KBM7 cells grown for four weeks post-treatment with alendronate (165 μM). (B) Sequencing-based identification of gene-trap insertion sites in alendronate-resistant cells. N=number of unique insertions within the stated gene locus. False discovery rate corrected (FDR) P-values for ATRAID=7.02×10⁻⁴⁵, PLCL1=1.02×10⁻⁰⁴, EPHB1=2.05×10⁻⁰⁴, SNTG1= 1.84×10⁻⁰³. P-values represent enrichment in alendronate-treated versus vehicle treated cells. (C) Schematic representation of structural features of human ATRAID protein and its mouse and frog orthologues. (D) Cell viability in wild-type control and ATRAID-deficient cells exogenously expressing or not expressing ATRAID cDNA. Cells were treated with alendronate (60 μM) and analyzed for cell viability. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. Error bars indicate the standard deviation for n=4 (biological replicates). N.S., not significant; *P < 0.05, student’s t-test. v2, variant 2 (NM_080592.3); v3, variant 3 (NM_001170795.1) of the ATRAID gene, respectively. (E) Chemical structures for nitrogen-containing bisphosphonates (N-BPs) or non-nitrogen-containing bisphosphonates (BP). KBM7 cell viability in ATRAID-deficient (ATRAID_GT1 and ATRAID_GT2) and control (wild-type) KBM7 cells upon treatment with nitrogen-containing bisphosphonates (N-BPs) or non-nitrogen-containing bisphosphonates (BP). All cells were treated with the indicated concentration of the indicated N-BP (alendronate, zoledronate), BP (etidronate, tiludronate) for 72 hours. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. All measurements were performed in quadruplicate (biological replicates). *P < 0.05, student’s t-test. (F) Immunoblots of cell lysates from ATRAID-deficient and ATRAID v3-reconstituted HEK-293T cells treated with the indicated dose of alendronate for 24 hours. Equal amounts of protein were loaded in each lane. This experiment was repeated three times (biological replicates) and was consistent all three times. *non-specific band.

ATRAID was named because it is a gene whose mRNA expression is strongly induced by the ligand all-trans retinoic acid (15). ATRAID is conserved in chordates and contains a signal peptide, Toll-like-receptor leucine rich repeat, EGF-like domain, and a transmembrane domain (Fig. 1C and fig. S1A) (19, 20). The alendronate resistance phenotype of ATRAID-deficient cells (ATRAID_GT1 (gene-trap1) and ATRAID_GT2 (gene-trap2) was reversed by the re-introduction of wild-type ATRAID splice variant 2 (v2) or splice variant 3 (v3) cDNA, which differ in their N-termini (Fig. 1D; fig. S1, A and B). To better understand the degree of alendronate resistance in ATRAID-deficient cells, we varied both cell number and drug concentration in the viability assay. ATRAID-deficient cells were resistant to alendronate over two to three orders of magnitude of drug concentration or cell number (fig. S1C). The growth of untreated wild-type and ATRAID-deficient cells didn’t differ (fig. S1D). Overexpression of full-length ATRAID (v2) sensitized cells to alendronate (fig. S1E). Lastly, ATRAID membrane targeting is required for the anti-proliferative effects of alendronate, as ATRAID-deficient cells complemented with full-length ATRAID (v2) were sensitive to the cytotoxic effects of alendronate, whereas those expressing the membrane truncated form remained resistant (fig. S1F). Taken together, these data establish ATRAID as a genetic factor required for the growth inhibitory effects of alendronate.

N-BPs are part of a larger class of compounds known as bisphosphonates (BPs) that contain two phosphate moieties each joined to a carbon atom by a carbon-phosphorus bond (21) (Fig. 1E). To determine whether the effects of ATRAID deficiency on alendronate resistance were specific to nitrogen-containing bisphosphonates, we tested the effect of several nitrogen-containing and non-nitrogen-containing bisphosphonates on wild-type and mutant ATRAID cells. ATRAID-deficient cells were resistant to the nitrogen-containing bisphosphonates, alendronate and zoledronate (ZOL), but were as sensitive to the non-nitrogen-containing bisphosphonates, etidronate and tiludronate, as control cells (Fig. 1E).

To determine whether ATRAID is required for the reduction in protein prenylation observed upon N-BP treatment, we monitored the prenylation of several proteins, including the heat shock protein DnaJ (Hsp40) homolog HDJ-2, and the Ras family GTPase Rap1a (22). Alendronate strongly inhibited prenylation of HDJ-2 and Rap1a in wild-type cells in a dose dependent manner and had much less of an effect on prenylation of these proteins in ATRAID-deficient cells (Fig. 1F). Furthermore, the inhibitory effect of alendronate on prenylation was rescued when ATRAID cDNA variants (v2 and v3) were introduced (fig. S1F). We observed inhibition of prenylation resistance at N-BP doses where we did not see PARP-1 cleavage in ATRAID deficient cells, suggesting that ATRAID can mediate the effect on prenylation independent of apoptosis (fig. S1G). Thus, these findings suggest ATRAID functions as a positive regulator upstream of FDPS.

ATRAID is required for organismal responses to nitrogen-containing bisphosphonates

To determine whether ATRAID modulates organismal responses to N-BPs we inactivated Atraid globally in mice (23). We confirmed deletion of Atraid exons 3–5 and determined that Atraid homozygous deleted Atraid^KO mice (labeled KO, −/−) are viable but their body weight is mildly reduced compared with litter-matched derived, wild-type controls (labeled as WT, +/+) (fig. S2, A to C). We confirmed that tail fibroblasts derived from Atraid^KO animals are resistant to the cytotoxic effects of alendronate (fig. S2, D and E). Before studying the effects of the N-BPs in the context of Atraid loss, we first characterized the basal role of Atraid in bones. We determined that Atraid mRNA expression was undetectable in the bones of Atraid^KO animals and that Atraid^KO mice had slightly smaller bones compared with litter-matched derived, wild-type control mice (fig. S2, F and G). To examine the effects of Atraid on bone structure, we performed micro-computed tomography (μCT) analysis (24). Atraid deficiency did not decrease either trabecular or cortical structural parameters (fig. S2, H and I; data file S1). We measured bone strength using three-point bending tests (25). Some measures, such as stiffness (Newtons/millimeter, N/mm) and post-yield displacement (a measure of bone fragility, in millimeters, mm) were decreased by Atraid deficiency, whereas others, such as yield load (the point where bone bending goes from elastic vs. plastic, in Newtons, N), were not significantly altered (fig. S2, J to L; data file S1, P > 0.05, student’s t-test).

Osteoclasts release degradation products from C-terminal telopeptides of type I collagen (CTX-I) from bone into blood (26), and CTX-I in serum was not significantly different in wild-type mice compared with Atraid^KO mice (fig. S2M; data file S1, P > 0.05, student’s t-test). Histomorphometric measures of osteoclast function including osteoclast surface per bone surface (Oc.S/BS) (27), as judged by Tartrate Resistant Acid Phosphatase (TRAP) staining (28), were also not statistically different (fig. S2N; data file S2). Consistent with a basal defect in osteoblast function (29), Atraid^KO mice have reduced serum circulating Gla-Osteocalcin [Gla-OC; the activated form of osteocalcin, incorporated in bone matrix (30)] and modestly reduced bone formation rate (BFR) as measured by double-labeling (27) (fig. S2, O and P; data file S1).

To test the effect of alendronate in a model that mimics menopausal bone loss, the most common indication for the N-BPs, we performed ovariectomies (OVX) on adult female mice (Fig. 2A) (31). When ovaries are removed from females, the changes in estrogen cause a reduction in bone density triggered by disruption of the balance of osteoblast and osteoclast functions. This loss of bone density can be alleviated by treatment with N-BPs (32). The magnitude of trabecular bone loss in WT and Atraid^KO sham mice four weeks after OVX is exemplified in the μCT 3D reconstruction of the femoral proximal metaphysis (Fig. 2B). Consistent with alendronate preventing bone loss (32), femoral cortical and trabecular structural parameters, including cortical thickness and area, bone volume/trabecular volume (%), and trabecular thickness, were increased by alendronate treatment of WT OVX mice (Fig. 2, C to F and data file S1; see WT OVX +/− alendronate). In contrast, alendronate had blunted effects in Atraid^KO OVX mice (Fig. 2, C to F and data file S1; see ATRAID^KO OVX +/− alendronate).

Fig. 2. Atraid is required for organismal responses to nitrogen-containing bisphosphonates.

(A) Schematic of mouse menopausal bone loss model – bilateral ovariectomy (OVX). Saline or 100 μg/kg/week alendronate was administered concurrent with OVX or a sham procedure. After four weeks, mice were euthanized and bones and serum were extracted and analyzed. (B) Representative μCT reconstructions of femoral trabecular bone from 4-month-old litter-matched derived, wild-type, Atraid WT (+/+), and KO (−/−), female mice that were either ovariectomized (OVX) or sham operated (Sham), treated with either vehicle (saline) (+OVX), or alendronate (+OVX+ALN) for four weeks. (C-F) Ovariectomized WT and Atraid KO mice and their bone microstructural responses to alendronate. Femur cortical (C, D) and trabeculae (E, F) regions were analyzed by μCT. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N=6–11 mice (3.5 month old) per group. *P < 0.01, ^# indicates 0.01<P<0.05, student’s t-test and red line indicates mean. (G-H) Ovariectomized WT and Atraid KO mice and their bone strength responses to alendronate. Stiffness (G) and yield load (H) were analyzed by three-point bending test. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N=6–11 mice per group. *P < 0.01, ^#0.01 < P < 0.05, N.S., indicates not significant, student’s t-test, and red line indicates mean.

The same patterns of alendronate resistance in Atraid^KO mice were observed for bone strength (Fig. 2, G and H). That is, alendronate increased bone strength as judged by stiffness and yield load in wild-type ovariectomized mice, but its effects were blunted in Atraid^KO matched cohorts (Fig. 2, G and H; data file S1). Taken together, these results suggest that Atraid is required for the beneficial effects of N-BPs in ovariectomized female mice.

To test an additional osteoporosis model, we examined senile (old age) osteoporosis using 18 month-old male WT and Atraid deficient mice (33). After treating these mice weekly with alendronate or saline for two months, we found similar results to those in our OVX study. That is, measures of bone density were increased by alendronate, but less so in the Atraid deficient mice (fig. S2, Q and R, data file S1). This further suggests Atraid is key for responses to N-BPs in vivo.

ATRAID is required cell-autonomously for N-BP inhibition of osteoclast function

Because N-BPs potentially affect osteoclasts and osteoblasts, we investigated whether Atraid deficiency would regulate the effects of alendronate in each cell type in our post-menopausal (OVX) and old-age (senile) osteoporosis models. Regarding osteoclasts, in wild-type mice both serum and bone histological markers of osteoclast function, CTX-I, and osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone surface (N.Oc/BS), respectively, were impaired by alendronate treatment (Fig. 3, A and B; fig. S3, A and B; data file S2) in both osteoporosis models. In contrast, in Atraid^KO mice, alendronate was less effective on osteoclasts in both osteoporosis models (Fig. 3, A and B; fig. S3, A and B; data file S2). That osteoclast number was reduced by N-BPs in wild-type mice is consistent with our cell viability measurements in non-osteoclasts and with previous literature (32).

Fig. 3. Atraid is required cell-autonomously for N-BP inhibition of osteoclast prenylation.

(A) CTX-I, a serum marker of osteoclast activity, was measured in WT and Atraid^KO ovariectomized mice with or without alendronate treatment by ELISA. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N=8–13 mice per group. *P < 0.05, student’s t-test. (B) Osteoclast histomorphometric responses in WT and Atraid^KO ovariectomized mice with or without alendronate treatment. Osteoclast surface to bone surface ratio (Oc.S/BS) was determined by Tartrate Acid Phosphatase (TRAP)-assay reactivity. Each circle represents an individual animal. Circles offset to the right represent unique animals with similar values to those of another animal (offset for visual clarity). N=5–7 mice per group. *P < 0.05, n.s. indicates not significant, student’s t-test, and red line indicates mean. (C) Quantitative PCR to examine mRNA expression of markers of osteoclast differentiation, Ctsk, Tnfrsf11a (RANK), Acp5 (TRAP), in wild-type (WT) and Atraid^KO M-CSF-expanded bone marrow macrophages (BMMs) differentiated with RANKL to osteoclasts. Expression is normalized to wild-type, undifferentiated BMM cells, using Actb and Rplp0 as control genes. Error bars represent the standard deviation of technical triplicate reactions. (D) Percent of Annexin-V positive cells after a 48 hour alendronate treatment of WT and Atraid^KO BMMs differentiated into osteoclasts. Annexin V staining was assessed using flow cytometry. Each circle represents osteoclasts derived from an individual animal (split for treatment with 0, 10 μM, 30 μM alendronate). Red line indicates mean. *P < 0.05, N.S. indicates not significant, student’s t-test. (E) Percent of Annexin-V positive cells after a 48 hour alendronate treatment (0, 30 μM, 80 μM) in wild-type and Atraid^KO differentiated RAW 264.7 osteoclasts. Annexin V staining was assessed using flow cytometry. Error bars represent the standard deviation of n=3 experiments (biological replicates), *P < 0.05, student’s t-test. (F) Immunoblots of cell lysates of RAW wild-type (WT) and Atraid^KO (KO) cells, and RAW 264.7-derived osteoclasts treated with alendronate for 48 hours. Top panel: immunoblot specific to the unprenylated version of Rap1a. Bottom panel: Gapdh, serving as a loading control. Alendronate concentrations were 0, 20 μM, 80 μM. (G) Representative image of a six-well dish co-culture of equal numbers of mouse primary osteoblasts and osteoclasts of the indicated genotypes with or without the indicated doses of alendronate (ALN) for four days. The experiment was performed three independent times with a similar result. Red staining reflects TRAP-assay reactivity. (H) Image analysis of the samples in (G). Error bars represent the standard deviation of n=3 independent images (technical replicates). *P < 0.01, N.S. indicates not significant, student’s t-test.

To provide insight into the effects of N-BPs on osteoblasts in our osteoporosis models, we measured BFR and mineral apposition rate (MAR) (27). Unlike BFR in which the rate is normalized by the amount of labeled bone surface, MAR is the rate of bone formation irrespective of how much of the bone is active (27). Alendronate did not affect trabecular MAR or BFR in either wild-type or Atraid^KO mice in either osteoporosis model (fig. S3, C and D; data file S2).

To determine whether Atraid is required in a cell autonomous manner for the N-BP-dependent effects on osteoclasts, we isolated M-CSF-expanded bone marrow macrophages (BMMs) from both WT and Atraid^KO mice, and differentiated these cells into osteoclasts following a standard protocol (34). Atraid^KO BMMs differentiated into osteoclasts as well as wild-type cells irrespective of treatment with alendronate, yet BMM-derived Atraid^KO osteoclasts were resistant to alendronate-induced apoptosis (Fig. 3, C and D). This suggests that Atraid is required cell autonomously in osteoclasts for the effects of N-BPs on cell number.

As an independent confirmation of our primary cell experiments, we generated Atraid knockout RAW 264.7 cells and differentiated them to osteoclasts (fig. S3E). RAW 264.7 cells are a robust, well-characterized murine macrophage cell line that can be differentiated to osteoclast-like cells using RANKL (35). We treated both RAW 264.7 cells and the RAW 264.7 cells differentiated into osteoclasts with alendronate, and found Atraid deficiency, as expected, conferred resistance to doses that induced apoptosis (Fig. 3E). Alendronate did not affect known markers of osteoclast differentiation in wild-type cells (Fig. 3C). Therefore, to pursue the mechanism of N-BPs on osteoclast function, we focused on prenylation. In alendronate-treated RAW 264.7 cells and osteoclasts differentiated from RAW 264.7 cells, we found that Atraid^KO cells were resistant to alendronate-induced inhibition of prenylation (Fig. 3F).

We assessed whether wild-type osteoblasts might sensitize Atraid-deficient osteoclasts to N-BPs. We cultured primary wild-type osteoblasts with either WT or Atraid-deficient primary osteoclasts and treated these co-cultures with alendronate or vehicle. As in the case of WT RAW 264.7 cells grown independently (Fig. 3, E and F), WT osteoclasts were more inhibited by alendronate than Atraid deficient osteoclasts despite the presence of WT osteoblasts in both cases (Fig. 3, G and H). In total, these findings support that Atraid is required for the cell-autonomous effects of N-BPs on osteoclasts.

Genetic factors involved in responses to nitrogen-containing bisphosphonates in patients

We sought an unbiased approach to determine what genes might be relevant in patients treated with N-BPs. We performed whole exome sequencing (WES) on two sets of patients taking N-BPs who experienced side effects: patients with osteoporosis who experienced atypical femoral fractures (AFF) (n = 27 patients), as well as patients with multiple myeloma or breast cancer patients who experienced osteonecrosis of the jaw (ONJ) (n = 8 patients) and 11 control patients taking N-BPs that didn’t experience AFF or ONJ (Fig. 4A and data file S3 for patient information). We also analyzed two published gene expression datasets involving patients who had taken N-BPs: patients with multiple myeloma who did or did not suffer ONJ when taking N-BPs (36), and patients with breast cancer with bone marrow disseminated tumor cells (DTC) which reoccurred or the patient died less than 1000 days vs. greater than 2500 days following initiation of zoledronate treatment (37) (Fig. 4A and data file S3). We then compared the patient data to three cell-based, genome-wide CRISPRi and CRISPRa screens we previously performed: alendronate and zoledronate CRISPRi and alendronate CRISPRa (12, 38) (data file S3). To identify genes involved in N-BP response across experimental paradigms we generated a Venn diagram to visualize the overlap of “hits”. In comparing the WES hits –genes that had the same rare coding variants (minor allele frequency < 0.05) in both patients with AFF and ONJ but not in controls – with our hits from our alendronate and zoledronate CRISPRi/a screens, we identified 64 genes in common including ATRAID, FDPS, and SLC37A3 (Fig. 4A). When comparing the WES hits with the gene expression hits, we identified 49 genes, whereas the CRISPRi/a and gene expression studies had 76 genes in common (Fig. 4A). Three genes, ATRAID, ATR, and ZBTB4 were statistically significant in all three data types (Fig. 4A) (data file S3, FDR corrected P < 0.05). Focusing on ATRAID specifically, we observed a ~50% decrease in ATRAID expression in the patients with DTC and ONJ that had their gene expression measured. By exome sequencing the patients with AFF and ONJ, in ATRAID we detected two rare variants – hereafter referred to as the ‘D5G/G32R variant’ – that were present together in 3 out of 35 patients with AFF and ONJ (2 out of 27 AFF; 1 out of 8 ONJ) (Fig. 4B).

Fig. 4. ATRAID as a potential genetic factor for altered responses to nitrogen-containing bisphosphonates in patients.

(A) Genome-wide studies of N-BP responsiveness in patients vs. cells. The outcomes considered from human studies involving N-BPs are: osteonecrosis of the jaw (ONJ), breast cancer bone marrow micrometastases [disseminated tumor cells (DTC)], and atypical femoral fractures (AFF). ATRAID, ATR, ZBTB4 are statistically significant hits (FDR corrected P < 0.05) in N-BP cell-based CRISPRi/a screening, differentially expressed in both gene expression datasets (ONJ and DTC), and possess rare multiple non-synonymous coding variants in AFF and ONJ cases but not controls. These three genes are visualized as the Venn diagram of overlap of lists of genes that met the following criteria: significant alendronate CRISPRi, zoledronate CRISPRi, or alendronate CRISPRa hits with absolute value of rho growth phenotype values ≥ 0.30 and P ≤ 0.05 (1335 out of 15828 genes) (12, 38); differentially expressed N-BP in ONJ + DTC (774 out of 18415 for ONJ (36) and 20492 for DTC (37); multiple coding variant(s) in AFF and ONJ cases and not controls (1252 out of 11659 genes) (data as part of this study). (B) Patient genetic data for ATRAID. Raw expression values were normalized to 1 to fit on a comparable Y axis. *P < 0.05, moderated t-test. N.S. indicates not significant. “X”-enriched refers to the fold-enrichment of the allele compared with a population with a similar genetic background as the cases. For example, for ATRAID, the D5G variant is present in 2 out of 27 AFF patients. Though this allele wasn’t detected in the 11 control samples, it is present in a population of European Americans (EA) and Asian Americans (AA) that is representative of the study population at a prevalence of 0.0131. Therefore, the D5G allele is (2/27) / 0.0131 = 5.66X enriched in cases compared to the EA/AA population. “v3” and “v2” refer to isoforms of the ATRAID gene. A simple binomial test was used to calculate the significance of each variant. * P < 0.05. (C) Quantitative PCR to examine ATRAID mRNA expression in wild-type, ATRAID-deficient, and low ATRAID expressing cells. Error bars represent the standard deviation of technical triplicate reactions. Expression was normalized to WT cells using RPLP0 and TBP as controls. * indicates P<0.05, student’s t-test. (D) Cell viability in wild-type, ATRAID-deficient, and low ATRAID expressing cells. Cells were treated with the indicated doses of alendronate and analyzed for cell viability. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. Error bars indicate the standard deviation for n=4 (biological replicates). N.S., not significant; * and ^# indicate P<0.05 for the indicated cell lines, student’s t-test. (E) Immunoblot (IB) of wild-type and D5G/G32R variant ATRAID-V5 tagged proteins. Mutant or wild-type ATRAID v2 and v3 were stably introduced into ATRAID-deficient HEK-293T cells. (F) Cell viability in wild-type vs. D5G/G32R variant cells. Cells were treated with the indicated doses of alendronate and analyzed for cell viability. Cell viability was determined by measuring cellular ATP and is expressed as a ratio of that compared with untreated cells. Error bars indicate the standard deviation for n=4 (biological replicates). N.S., not significant; *P < 0.05, student’s t-test.

We sought to determine the functional relevance of decreased ATRAID mRNA expression and the ATRAID D5G/G32R variant, both of which are associated with bad outcomes of N-BP treatment (Fig. 4, A and B). To test the former, we expressed ATRAID mRNA at sub-endogenous quantities in ATRAID deficient cells such that the expression was similar to the reduced expression seen in patients that experienced ONJ or DTC (~50% compared to wild-type controls) (Fig. 4C). We refer to these ATRAID partially restored cells as ‘ATRAID_{low expr.}’. ATRAID-deficient cells conferred resistance to alendronate as expected, whereas ATRAID_{low expr.} cells were hypersensitized to alendronate (Fig. 4D). Similarly, the ATRAID D5G/G32R variant, which we identified in both the patients with AFF and ONJ, conferred hypersensitivity to alendronate compared with wild-type ATRAID (Fig. 4, E and F). Taken together, this suggests that bad patient outcomes might reflect cellular hyper-response to N-BPs. In total, these findings support the importance of ATRAID in bisphosphonate responsiveness in humans.

DISCUSSION

This work focused on the physiological impact of ATRAID as a positive regulator genetically upstream of FDPS. Here we use prenylation as an output of FDPS function. Recently, we linked FDPS to DNA synthesis and damage (39). This was intriguing in light of earlier studies in the context of ONJ where N-BPs regulated p63 – a well-known mediator of DNA damage (40) – in a mevalonate pathway-dependent manner (41). Considering that each of the three top genes from the patient analysis, ATRAID, ATR, and ZBTB4, are involved in p53 responses – a better known p63-related mediator of DNA damage (42–45) – it will be interesting to determine whether these genes mediate their effects on p53/p63 signaling via FDPS.

The molecular effects of N-BPs on FDPS require the transporter, SLC37A3 (12). Interestingly, the SLC37A family member (46), SLC37A2, is mutated in dogs and gives rise to a bone overgrowth phenotype that resembles the human disease Caffey syndrome (47, 48). This phenotype is particularly interesting because it suggests that natural ligands or drugs that inhibit the SLC37A family might phenocopy N-BP treatment in increasing bone density.

ATRAID binds NELL-1, a secreted protein that promotes bone mineralization in mice and potentiates osteoblast differentiation in an ATRAID-dependent manner (49, 50). It is also notable that NELL-1 is in pre-clinical testing for the treatment of osteoporosis (51). In future studies, it will be interesting to determine whether NELL-1 affects the responses to N-BPs we observe upon manipulating ATRAID. NELL-1 has a related family member, NELL-2. This family member has been the subject of high profile studies in the field of axon guidance (52). It is unknown whether ATRAID signals to NELL-2 and if so what role it may play in the brain.

There are several limitations of our study. 1) Because we used a global knockout strategy with Atraid, we can’t definitively conclude it is required in vivo in osteoclasts – the target cell type for the N-BPs; 2) We identified ATRAID in screening in leukemia cells, not in osteoclasts. Therefore, there it is possible a screen in a cell type more relevant to the N-BPs would yield additional genes important to bone; 3) There are relatively modest numbers of DNA samples in existence from ONJ and AFF patients. More samples need to be collected and analyzed to further test our findings as identifying those patients who might experience these consequences when taking N-BPs is of paramount importance.

MATERIALS AND METHODS

Study design.

The objective of this study was to identify and subsequently characterize factors involved in the on- and off-target effects of the osteoporosis drugs, nitrogen-containing bisphosphonates (N-BPs). To address this, we performed a genome-wide haploid cell screen and identified the gene ATRAID. To assess the cellular role of ATRAID in the response to N-BPs, we treated a variety of cell lines including human 293T and KBM7 cells, murine macrophage RAW 264.7 cells differentiated into osteoclasts and primary cells derived from mice, with the N-BPs (including alendronate and zoledronate) or other drugs and assessed cell viability/growth/fitness by measuring cellular ATP, as well as protein prenylation by immunoblot. These analyses established that ATRAID is required for the cellular responses to N-BPs. We investigated the in vivo role of ATRAID by utilizing two mouse models of osteoporosis (ovariectomies on 3.5-month old female mice as a model of post-menopausal osteoporosis (OVX), and 18-month old male mice as a model for senile osteoporosis) treated with alendronate. The effects on wildtype and Atraid^KO mice were assessed by profiling bone structure using micro-computed tomography (μCT) (OVX, senile: n = 6–11, n = 5–8), strength using a three-point bending assay (OVX: n = 6–11), histomorphometry using TRAP staining and double-labeling (OVX, senile: n = 5–7, n = 4–7), and serum bone proteins using ELISA (OVX, n = 8–13). These analyses established that ATRAID is required for the organismal responses to N-BPs. To translate our findings to humans, we integrated clinical and unbiased genome-scale molecular data in patients treated with N-BPs, including patients that experienced atypical femoral fractures or osteonecrosis of the jaw while being treated with N-BPs. These analyses established that ATRAID is potentially important for N-BP responses in humans. For our animal studies, mice were randomized to treatment groups, and subsequent analyses were blinded to the extent possible. All experiments involving mice were performed with protocols approved by the Harvard and Washington University Animal Studies Committees. The details of study design, sample sizes, experimental replicates, and statistics are provided in the corresponding figures, figure legends, data files, and Material and Methods.

Statistical analysis.

Unless otherwise specified, group means were compared by one-tailed student’s t test for unpaired samples. Data on repeated measures were analyzed by ANOVA, followed by a post-hoc multiple Holm–Sidak method t-test. All data are expressed as the mean ± s.d with numbers of samples indicated in figure legends. P values are indicated in each figure legend, and values less than 0.05 were considered significant (alpha) with ≥ 80% power, unless indicated otherwise. We estimated the cohort sizes we would need for this study based on our prior study which involved a similar experimental paradigm in using bisphosphonates and the ovariectomy (OVX) osteoporosis model in BL/6 mice (32). All code used to generate statistics and correlations for this project can be found at https://github.com/tim-peterson/ATRAID (DOI: 10.5281/zenodo.3739576).

Supplementary Material

Data File S3

Data file S3. Gene expression, sequencing, and growth phenotype data for ONJ, DTC, AFF and CRISPRi and CRISPRa studies.

Data File S2

Data file S2. Statistics for bone histomorphometry and serum bone proteins in ovariectomized and senile wildtype and Atraid^KO animals treated with alendronate.

Data File S1

Data file S1. Statistics for Atraid^KO mice basal characterization, and statistics for bone structure, strength of ovariectomized wildtype and Atraid^KO animals treated with alendronate.

Table S1

Table S1. Results of haploid genomic screen for genes required for the response to alendronate.

ATRAID_Supp_text_and_figures

Fig. S1. ATRAID is required for the cellular responses to nitrogen-containing bisphosphonates.

Fig. S2. Generation and skeletal characterization of Atraid^KO mice.

Fig. S3. Atraid is required cell-autonomously for the effects of N-BP on osteoclasts in two models of osteoporosis.

Data and materials availability:

All data associated with this study are present in the paper, the Supplementary Materials, or will be available at NCBI BioProject ID: PRJNA624650. Shared reagents are subject to a materials transfer agreement.