O-GlcNAcylation of MITF regulates its activity and CDK4/6 inhibitor resistance in breast cancer

Abstract Cyclin-dependent kinases 4 and 6 (CDK4/6) play a pivotal role in cell cycle and cancer development. Targeting CDK4/6 has demonstrated promising effects against breast cancer. However, resistance to CDK4/6 inhibitors (CDK4/6i), such as palbociclib, remains a substantial challenge in clinical settings. Using high-throughput combinatorial drug screening and genomic sequencing, we found that the microphthalmia-associated transcription factor (MITF) is activated via O-GlcNAcylation by O-GlcNAc transferase (OGT) in palbociclib-resistant breast cancer cells and tumors; O-GlcNAcylation of MITF at Serine 49 enhanced its interaction with importin α/β, thus promoting its translocation to nuclei, where it suppressed palbociclib-induced senescence; inhibition of MITF or its O-GlcNAcylation re-sensitized resistant cells to palbociclib. Remarkably, clinical studies confirmed the activation of MITF in tumors from patients who are palbociclib-resistant or undergoing palbociclib treatment. Collectively, our studies shed light on a novel mechanism regulating palbociclib-resistance, and present clinical evidence for developing therapeutic approaches to treat CDK4/6i-resistant breast cancer patients. Significance: This work not only identifies a novel mechanism regulating MITF activity and resistance to CDK4/6 inhibitors in breast cancer cells, but also provides clinical evidence supporting the development of therapeutic approaches to treat CDK4/6i resistant breast cancer patients by targeting MITF.


INTRODUCTION
The cyclin-dependent kinase (CDK) 4/6 are serine/threonine kinases that are associated with D-type cyclins, such as Cyclin D1, to promote the G1-S phase transition by phosphorylating retinoblastoma protein (RB), thus resulting in activation of E2F transcription factors that facilitate cell cycle progression 1,2,3,4 .CDK4/6 proteins are frequently upregulated and activated in a variety of cancers 5,6,7,8 , making them attractive targets for therapeutic intervention.As a result, inhibiting CDK4/6 has emerged as a promising strategy for treating cancer.Presently, three CDK4/6 inhibitors (CDK4/6i), palbociclib, ribociclib, and abemaciclib, have gained FDA approval for the treatment of metastatic ER-positive, HER2-negative breast cancer in combination with hormone therapy 9,10,11 .Although the majority of patients initially respond well to CDK4/6i-based therapy and delay progression, the development of resistance poses a signi cant challenge to effective clinical management and long-term survival 12 .Although recent studies have implicated several resistant mechanisms, including mutations in RB1 or FAT1, and activation of CDK4/6, CDK2, PI3K/AKT, RAS/ERK, or STAT3 13,14,15,16,17,18,19 , treatment strategies to overcome CDK4/6i resistance remain an unmet clinical need.
Microphthalmia-associated transcription factor (MITF) belongs to the basic-helix-loop-helix-leucine zipper (bHLH-ZIP) family, constituting the MiT/TFE transcription factor family in conjunction with TFEB, TFEC, and TFE3 20 .These MiT/TFE members regulate downstream gene expression by binding to the E-box motif, characterized by the palindromic canonical sequence of CACGTG 21 .MITF encompasses several isoforms with distinct amino termini, including MITF-A, MITF-C, MIT-E/D, MITF-B, MITF-C, MITF-H, and MITF-M 22 .Unlike other MITF isoforms, MITF-M is the predominant isoform expressed in melanocytes and melanoma cells, making it a potential biomarker of melanoma 23 .Under typical growth conditions, MITF-M is constitutively retained within nuclei 24 .In contrast, MITF-A, along with other isoforms, features an N-terminal 1B1b domain with speci c residues facilitating their cytoplasmic localization.This localization arises due to cytoplasmic retention after phosphorylation of S173 by TAK1 in osteoclasts 25 , or via phosphorylation by mTORC1 and the interaction with the RAG GTPases at the surface of the lysosome 26 .Notably, exogenous expression of MITF-A led to predominantly cytoplasmic localization, while mutation of conserved residues Q62 and L63 within exon B1b prevents cytoplasmic retention of MITF-A 27 .The distinct cellular localization of MITF-A implies potential divergent functionality compared to MITF-M in cells.So far, compared to MITF-M, our understanding of the regulatory mechanism governing MITF-A in cancer cells remains limited 23 .Even less is known about the physiological roles of MITF-A and other isoforms in response to drug treatments and drug resistance.
O-GlcNAcylation is a unique type of posttranslational modi cation that involves the addition of a single O-link N-acetylglucosamine (O-GlcNAc) sugar moiety to the serine (Ser) or threonine (Thr) residues of target proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAase (OGA) 28,29,30 .Dysregulation of O-GlcNAcylation has been linked to various diseases, such as diabetes, neurodegenerative disorders, and cancers 31,32,33,34 .Emerging evidence has indicated that aberrant O-GlcNAcylation may contribute to tumorigenesis, promote drug resistance, and in uence the immune response by altering protein stability, localization, or interactions 35,36,37,38 .However, the role of O-GlcNAc signaling in the regulation of CDK4/6i resistance remains unclear.
In this study, we performed an unbiased quantitative high-throughput combinatorial screening (qHTCS) to identify compounds capable of overcoming resistance to CDK4/6i in breast cancer cells.Through this screening, we identi ed ML329, a MITF inhibitor, which effectively restored sensitivity to palbociclibresistant cells, both in vitro and in vivo.In-depth mechanistic analyses revealed that palbociclib-resistant breast cancer cells exhibited elevated levels of MITF-A and its O-GlcNAcylation.Notably, O-GlcNAcylation of MITF-A at Serine 49 (S49) by O-GlcNAc transferase (OGT) facilitated its interaction with importin α/β, thereby facilitating nuclear translocation.Within the nucleus, MITF-A curtailed palbociclib-induced senescence, ultimately driving resistance.Importantly, inhibition of MITF or its O-GlcNAcylation sensitized resistant cells to palbociclib both in vitro and in vivo.Remarkably, this previously unappreciated O-GlcNAcylation-MITF-mediated mechanism was corroborated in resistant patient-derived xenograft (PDX) lines, as well as tumor samples from patients who received CDK4/6i treatment.Collectively, these ndings not only shed light on an innovative regulatory mechanism governing MITF's activity and its role in palbociclib resistance but also inform the development of a novel therapeutic strategy for effectively managing palbociclib-resistant breast cancer patients.

Cell culture
Human breast cancer cell lines MCF-7 and T-47D were purchased from ATCC and cultured in DMEM medium supplemented with 10% FBS at 37°C with 5% CO 2 .The palbociclib-resistant cell lines MCF-7 PR and T-47D PR were generated by exposing them to palbociclib with gradually increasing concentration over 6 months.Only early-passage (< 10 passages) resistance cells were used for the study.
Quantitative high-throughput combinatorial screen (qHTCS) Compound screen experiments were performed in two rounds as described previously 39 .MCF-7 and MCF-7 PR cells were used against two compound libraries including MIPE (Mechanism Interrogation Plate) and NPC (NIH Pharmaceutical Collection) library.Brie y, MCF-7 and MCF-7 PR cells (1,000 cells/well) were plated in 1,536 wells and incubated for 24 hr.In the rst screen, drugs were tested at 8 different concentrations from 0.8 nmol/L to 46 µmol/L by serial dilution (1:3).After incubation for 72 hr, a total of 120 compounds that e ciently inhibited the proliferation of both MCF-7 and MCF-7 PR cells were identi ed.To further identify compounds that have synergy with palbociclib in MCF-7 PR cells, the top 20 compounds were selected for the second screen together with palbociclib.These 20 compounds were screened at 10 different concentrations in combination with 10 different concentrations of palbociclib.
Then 4-µL/well ATP content cell viability assay reagent (Promega, G7570) was added into each well and incubated for 15 min followed by detection of cell viability.Through the second screen, compounds that exhibited the increased cytotoxicity of palbociclib in MCF-7 PR cells were identi ed.The primary screen data and curve tting were analyzed using software developed at the NIH Chemical Genomics Center (NCGC).

RNA-seq analysis
Total RNA from cells MCF- Immuno uorescence assay and Sulforhodamine B (SRB) assay The immuno uorescence assay was performed as we described previously 40 , and the SRB assay was performed as described previously 41 .

Senescence-associated β-galactosidase assay
The senescence-associated β-galactosidase staining was conducted in MCF-7 PR cells with the kit (Cell signaling technology, #9860) according to the manufacturer's instructions.

Biotin pull-down assay
The oligonucleotides containing MITF binding motif CATGTG region were used for Biotin-pull down assays.Brie y, cells were lysed and nuclear fraction was separated, and incubated with biotin-labeled oligos and 20 µl of streptavidin beads overnight.Beads were then washed three times and subjected to SDS-PAGE and blotted with antibodies as indicated.

Co-immunoprecipitation
Cells were lysed with Co-IP buffer containing 20 mM Tris-HCl at pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10 mM NaF, and protease inhibitor cocktail (Roche) on ice for 30 min, followed by sonication 10 sec for three times.After centrifuge, the supernatant was collected and incubated with protein A/G beads coupled with the antibody against indicated proteins at 4℃ overnight.The beads were washed three times and analyzed by Western blot.For FLAG IP, cell lysates were incubated with Anti-FLAG-M2 A nity beads overnight and IPs were analyzed by Western blot.
Mass spectrometry MCF-7 PR cells were transfected with FLAG-vector or FLAG-MITF for 48 hr, then cells were harvested for immune precipitation (IP).Eluted proteins after immunoprecipitation were then resolved by SDS-PAGE followed by Commassie blue staining.Corresponding gel bands were excised and in-gel digestion with trypsin, with the digests analyzed by a nanoUPLC-MS/MS system as previously described 42 .In brief, peptides were analyzed with nanoAcquity UPLC coupled with an Orbitrap Lumos mass spectrometer in the EThcD mode.The resulting data les were processed with Proteome Dicoverer 2.4 (Thermo Fisher Scienti c), by database searching against the customized human MITF protein database.Manual con rmation was performed for the potential O-GlcNAc sites assigned.Mass Data was listed in Supplementary Table 3 and is available via ProteomeXchange with identi ers PXD042763 and PXD042838.

In vitro O-GlcNAcylation assays
Recombinant His-OGT protein (1 mg) was incubated with 2 mg of recombinant GST-MITF or its mutant GST-MITF-S49A in 60 µL reaction volume (50 mM Tris-HCl, 12.5 mM MgCl2, 2 mM UDP-GlcNAc 1 mM DTT, pH 7.5) at 37℃ for 4 hr.Samples were separated by SDS-PAGE and subjected to immunoblotting with the indicated antibodies.
GST pull-down assays GST-MITF fusion protein was puri ed from E. coli and immobilized on glutathione Sepharose 4B columns (GE Healthcare).HEK293T cells transfected with the indicated plasmids were lysed in NETN buffer with protease inhibitor cocktail (Sigma-Aldrich, USA) and incubated with Sepharose beads immobilized with the indicated GST-tagged proteins at 4 ℃ overnight.After washing the beads were boiled in 60 µL 2×SDS loading buffer and subjected to immunoblotting with the indicated antibodies.

Animal experiments
Six-week-old female BALB/c athymic nude mice were purchased from Jackson Laboratory.4×10 6 MCF-7 PR or MCF-7 PR MITF-depleted cells were subcutaneously injected into the dorsal ank of each mouse.
When the tumor volume reached about 100 mm 3 , all mice were randomized into indicated experiment groups.Palbociclib was administrated intraperitoneally at 25 mg/kg every 2 days for 2 weeks.Saline was used as the control.Relative tumor volumes were calculated with the formula: V = A×(B 2 )/2, where A and B represent the length and width of each tumor, respectively.For immunohistochemistry (IHC) analysis, tumor samples were sliced and conducted standard IHC staining procedures, and DAB staining was done utilizing SuperPicture™ Polymer Detection Kit (Thermo Fisher Scienti c) according to the manufacturer's instructions.All animal studies were carried out by the National Institutes of Health regulation concerning the care and use of experimental animals and with the approval of the Institutional Animal Care and Use Committees of George Washington University.
Identi cation and analysis of MITF signature geneset in the NeoPalAna trial MITF signature geneset was created by overlapping proteins that interact with MITF (mass-spec analysis) and genes that are known to be associated with MITF function by PathwayNet 43 , and then overlap genes of these two groups were selected and named as MITF signature geneset (Supplementary Table 3).MITF signature geneset was then used as bait to examine the microarray gene expression data obtained from the tumor biopsies in the NeoPalAna trial 44 .Log 2 -normalized expression data from baseline samples were used for the analysis.The gene set enrichment analysis (GSEA) software was used for calculating enrichment scores.
Statistical analysis GraphPad Prism 9.0 software was utilized for all data analyses.Data were represented as mean ± SEM as indicated in the gure legends.Statistical analysis was performed using one-way ANOVA or Student's t-test.Differences were considered statistically signi cant at a p-value of less than 0.05.

RESULTS qHTCS identi es MITF inhibitor ML329 that overcomes palbociclib resistance
To identify the effective therapeutic strategies to overcome palbociclib-resistance in breast cancer cells, we generated two palbociclib-resistant (PR) breast cancer cells (MCF-7 PR and T-47D PR) by continuously treating cells with palbociclib using approaches as we described previously 45 (Supp Fig. 1A-1B).The MCF-7 PR cells also exhibited resistance to the other two CDK4/6i, ribociclib and abemacicilib (Supp Fig. 1C-1D).Using MCF-7 and MCF-7 PR cells, we conducted a two-round qHTCS with MIPE (Mechanism Interrogation Plate) and NPC (NCATS Pharmaceutical Collection) small molecular compound libraries.
From the rst-round screening, we identi ed a total of 120 compounds that e ciently suppressed the proliferation of both MCF-7 and MCF-7 PR cells.To identify compounds that synergize with palbociclib in MCF-7 PR cells, we selected the 20 most active compounds against MCF-7 PR cells for the second-round screen together with palbociclib (Fig. 1A).In the second screening, a MITF inhibitor called ML329 was found to be one of the top hits that could resensitize MCF-7 PR cells to palbociclib (Fig. 1A).To validate the synergistic effects of ML329 with palbociclib, MCF-7 PR and T-47D PR cells were treated with a combination of ML329 and palbociclib for 72 hr and cell viability was measured and calculated using Combene t as described 46 .Signi cantly, the combined treatment of ML329 with palbociclib inhibited the growth of both resistant cells (Fig. 1B).To precisely measure the synergy, we calculated the combination index (CI), a quantitative measure of the interaction between two drugs (synergism: CI < 1; additive effect: CI = 1; and antagonism: CI > 1) 47 .Remarkably, the combination of palbociclib and ML329 exhibited a great synergy in both resistant cells (Supp Fig. 1E-1F).
To test whether combinatorial treatment affects cell cycle progression, we examined the proliferation of resistant cells by using the Edu incorporation assay.Palbociclib or ML329 alone had little or limited impact on cell cycle progression as indicated by S phase population of MCF-7 PR cells, however, the combinatorial treatment of ML329 and palbociclib dramatically reduced the S phase population of MCF-7 PR cells, suggesting that combination but not individual drug treatments lead to a cell cycle arrest before S phase (Fig. 1C).In agreement with these synergistic effects, the combinatorial treatment led to a substantial reduction in the colony formation capacity of both resistant cells (Fig. 1D-1E) and resensitized resistant MCF-7 PR tumors to palbociclib in vivo (Fig. 1F-1G and Supp Fig. 1G).Collectively, these results suggest that MITF inhibitor ML329 is able to overcome palbociclib resistance in breast cancer cells.

Inhibition of MITF overcomes palbociclib resistance by activating the senescence pathway in breast cancer cells
We next investigated how MITF regulates palbociclib resistance.Given that the MITF gene transcribes multiple isoforms 48 , we rst set to determine which MITF isoform is enriched in resistant cells.Using RT-PCR, we found that MITF-A is the prevalent isoform highly expressed in both MCF-7 PR and T-47D PR cells compared to their sensitive counterparts (Supp Fig. 2A) (We refer MITF as MITF-A in the following text).Both RNA-seq and Western blot analyses indicated that MITF mRNA and protein expressions were signi cantly increased in resistant cells compared to sensitive cells (Fig. 2A-2B).Interestingly, suppression of MITF by small hairpin RNA (shRNA) re-sensitized both resistant cells to palbociclib as indicated by cell survival and colony formation assays (Fig. 2C and Supp Fig. 2B-2D).Consistently, depletion of MITF by shRNA together with palbociclib treatment resulted in a signi cant decrease of phosphorylated Rb, while simultaneously leading to a remarkable increase of p21 level compared to treatment with palbociclib alone (Fig. 2D and Supp Fig. 2E), indicating that MITF depletion together with palbociclib leads to cell cycle arrest of resistant cells.Similar results were observed when MITF was inhibited by ML329 treatment in both resistant cells (Supp Fig. 2F).In agreement with these in vitro results, inhibition of MITF by shRNA re-sensitized MCF-7 PR tumors to palbociclib in vivo (Fig. 2E-2F and Supp Fig. 2G).Thus, MITF appears to play an important role in regulating palbociclib resistance in CDK4/6i resistant breast cancer cells.
Since MITF inhibition together with palbociclib increased expression of p21 (Fig. 2D), a marker of senescence 49 , we hypothesized that depletion of MITF re-sensitizes PR cells to palbociclib by inducing senescence.Indeed, inhibition of MITF resulted in a signi cant enrichment of the senescence-associated secretory phenotype (SASP) geneset in MCF-7 PR cells (Fig. 2G).Meanwhile, RNA-seq analysis indicated that the expressions of multiple SASP genes (CXCL8, IL-6, CDKN1A, IGFBP7, CXCL12, SERPINE1, CCL-2, IGFBP2, and MMP-10) were highly induced in MITF-depleted cells (Fig. 2H).These up-regulated genes upon MITF depletion were further con rmed by qPCR analysis (Fig. 2I).Consistently, inhibition of MITF alone increased the number of cells with positive staining of senescence-associated β-galactosidase (SAβ-Gal); and inhibition of MITF by siRNA or ML329 in combination with palbociclib further augmented the cell population undergoing senescence in MCF-7 PR cells (Fig. 2J-2K).Additionally, co-administration of ML329 and palbociclib did not induce apoptosis as indicated by the absence of cleaved PARP (Supp Fig. 2H).Thus, MITF inhibition overcomes palbociclib resistance by activating senescence in resistant breast cancer cells.

OGT interacts with MITF and promotes its nuclear translocation
To determine how MITF regulates palbociclib resistance, we rst examined the subcellular localization of MITF in sensitive and resistant cells.Surprisingly, unlike sensitive cells in which MITF predominantly resided in the cytoplasm, MITF primarily localized in nuclei in both resistant cells (Fig. 3A).Previous studies reported that MITF is phosphorylated by mTORC1 and this phosphorylation leads to its interaction with 14-3-3 proteins and cytoplasmic retention 26, 50 .In agreement with this observation, the interaction of MITF with 14-3-3 was decreased in both resistant cells compared to their sensitive counterparts (Supp Fig. 3A-3B).Additionally, the association between MITF and 14-3-3 was diminished upon Torin1 (an mTORC1 inhibitor) treatment (Supp Fig. 3A-3B).Thus, MITF primarily localizes within nuclei in resistant cells compared to sensitive cells.
To explore the mechanism that supersedes cytoplasmic retention of MITF in resistant cells, we conducted a mass spectrometry analysis to identify MITF-associated proteins in MCF-7 PR cells.Notably, we found that OGT, the O-linked N-acetylglucosamine (GlcNAc) transferase, was signi cantly enriched in MITF immunoprecipitated fraction (Fig. 3B).Co-immunoprecipitation (Co-IP) assay validated the association between endogenous MITF and OGT, and vice versa (Fig. 3C).Consistently, OGT directly O-GlcNAcylated MITF in vitro (Fig. 3D).Given that OGT was highly expressed in resistant cells compared to sensitive cells (Supp Fig. 3C), we postulated that OGT might regulate MITF activity through O-GlcNAcylation in resistant cells.Indeed, overexpression or knockdown of OGT signi cantly increased or decreased O-GlcNAcylation of MITF respectively (Fig. 3E-3F), and inhibition of OGT did not affect MITF protein levels but signi cantly reduced its nuclear accumulation in MCF-7 PR cells (Fig. 3G and Supp Fig. 3D-3E).In agreement with these data, OGT depletion or treatment with the OGT inhibitor OSMI-1 signi cantly increased the interaction between MITF and 14-3-3 (Fig. 3H-3I), whereas this interaction was reduced upon PUGNAc (an OGA inhibitor) treatment (Fig. 3J).Moreover, overexpression of OGT promoted the translocation of MITF from the cytoplasm into the nucleus in sensitive MCF-7 cells (Supp Fig. 3F).We next tested whether or not O-GlcNAcylation of MITF in uences its interaction with its cognate DNA binding sequence.To this end, we synthesized an oligonucleotide containing the MITF binding motif (E-box), and tagged it with biotin, and then mixed it with nuclear lysates from cells.The results indicated that OGT depletion signi cantly decreased the interaction of MITF with DNA binding motifs (Fig. 3K).Accordingly, depletion of OGT also sensitized both resistant cells to palbociclib (Fig. 3L-3M).Collectively, the above results strongly indicate that OGT directly interacts with and O-GlcNAcylates MITF, which in turn prevents its interaction with 14-3-3, promoting its nuclear translocation and its a nity to its DNA binding motif.

O-GlcNAcylation of MITF at S49 is required for nuclear accumulation and resistance
To further investigate how O-GlcNAcylation regulates MITF activity, we conducted a mass spectrometry assay to identify the speci c O-Glycosylation site of MITF.The analysis found that O-GlcNAcylation of MITF predominantly occurred on a peptide that spans residues 34-56, and S49 was the most likely glycosylation site (Fig. 4A).To exclude the possibility of GlcNAc modi cations on the MITF peptide was resulted from other Ser/Thr sites rather than S49 site, we mutated S49 as well as other potential O-GlcNAcylation sites from serine to alanine.Interestingly, the O-GlcNAc level of MITF was markedly reduced only in S49A mutant in vitro and in vivo (Fig. 4B-4C), suggesting that S49 is the primary O-GlcNAc site of MITF.Importantly, sequence comparison analysis showed that MITF S49 was conserved across various species (Supp Fig. 4A).
We next investigated how S49 O-GlcNAcylation regulates MITF activity.Interestingly, the mutant MITF (S49A) exhibited reduced levels of O-GlcNAc and increased interaction with 14-3-3 compared to wild-type (WT) MITF (Fig. 4D).Moreover, both nuclear fractionation and IF assays indicated that WT MITF but not MITF(S49A) displayed an increased nuclear localization when O-GlcNAcylation was enhanced by OGT overexpression (Fig. 4E-4F).Consistently, WT MITF displayed a signi cantly augmented binding to the conserved DNA binding motif of MITF in comparison to the S49A mutant (Fig. 4G).Depletion of MITF in MCF-7 PR cells reduced the expression of MITF targeting genes CCND1, BIRC1, and CCNB1, which was restored by the expression of WT MITF but not the S49A mutant (Fig. 4H).The above results indicated a critical role of O-GlcNAcylation of MITF at S49 in the regulation of its nuclear localization and transcriptional activity.
We next investigated how O-GlcNAcylation at S49 regulates the response of resistant cells to palbociclib.Interestingly, depletion of MITF re-sensitized MCF-7 PR cells to palbociclib, which was rescued by reconstitution of WT MITF but not S49A mutant (Fig. 4I and Supp Fig. 4B).Using xenografted tumor models, we also found that reintroduction of WT MITF but not S49A mutant in MITF depleted-MCF-7 PR cells restored resistance to palbociclib.Thus, O-GlcNAcylation at S49 of MITF plays a critical role in the regulation of palbociclib resistance in breast cancer cells.

O-GlcNAcylation within the nuclear localization signal (NLS) promotes the interaction of MITF with importin α/β
We next explored how O-GlcNAcylation regulates the nuclear translocation of MITF in resistant cells.MITF contains two putative nuclear localization signal (NLS) regions (Fig. 5A).Depletion of the rst NLS, but not the second NLS, hindered the nuclear accumulation of MITF in MCF-7 PR cells (Fig. 5B), indicating that the rst NLS is responsible for its nuclear translocation.Since O-GlcNAcylation site S49 is localized within the rst NLS, we assumed that O-GlcNAcylation of MITF regulates its nuclear translocation by affecting its interaction with importin α/β, which are membrane-associated proteins playing a critical role in regulating the transport of proteins between cytosol and nuclei 51 .To identify which importin is involved in the regulation of MITF nuclear translocation, we examined MITF localization by IF in cells with depletion of various importins using siRNAs.Depletion of importin α4, α7, and α8 appeared to compromise MITF nuclear localization and the knockdown of these three importin proteins together led to the robust decrease of nuclear translocation of MITF (Fig. 5C and Supp Fig. 5A), suggesting that importin α4, α7, and α8 are essential for MITF nuclear translocation.Moreover, we also observed that suppression of importin β reduced the nuclear translocation of MITF (Supp Fig. 5B).These ndings prompted us to test whether O-GlcNAcylation regulates the nuclear translocation of MITF by in uencing the association of MITF with importin α/β.Intriguingly, inhibition of OGT in MCF-7 PR cells dramatically reduced the interactions of MITF with importins α4, α7, α8, as well as importin β (Fig. 5D-5E).
To further study how O-GlcNAcylation regulates the interaction between MITF and these importins, we generated O-GlcNAcylated MITF by in vitro O-GlcNAcylation assay.As shown in Fig. 5F, O-GlcNAcylated MITF displayed an enhanced interaction with these importins compared to non-O-GlcNAcylated MITF by GST pull-down assay, suggesting that O-GlcNAcylation promotes the recognition of the NLS of MITF by these importins.Moreover, WT MITF exhibited a stronger interaction with importins α4, α7, and α8 than S49A mutant, and increasing O-GlcNAcylation by PUGNAc treatment further enhanced the association of WT MITF but not S49A mutant with importins α4, α7, and α8 (Fig. 5G-5I).The aforementioned results strongly suggest that importin α/β functions as a sensor to recognize the O-GlcNAcylated NLS of MITF and facilitate its nuclear translocation.

O-GlcNAcylation of MITF contributes to palbociclib resistance by regulating senescence signaling
To investigate how MITF regulates palbociclib resistance in breast cancer cells, we performed MITF ChIPseq in MCF-7 and MCF-7 PR cells.Interestingly, the ChIP-seq identi ed over 3000 MITF-occupied sites located in both inter and intragenic regions throughout the genome in MCF-7 PR cells (Fig. 6A).In comparison, MITF exhibited a relatively higher binding intensity in MCF-7 PR cells compared to sensitive cells (Fig. 6B).From ChIP-seq analysis, we observed that MITF displayed a stronger a nity towards the promoter region of four SASP factors, SERPINE1, IL-6, CSF1, and PTGER2 in MCF-7 PR cells compared to MCF-7 cells (Fig. 6C).All these four genes have been reported to positively regulate senescence individually 52,53,54 .We assumed that MITF may suppress the expression of these genes in resistant cells.Indeed, ChIP-qPCR indicated that MITF speci cally bound to the promoter region of these four genes (TYR gene was used as a positive control) (Fig. 6D), and depletion of MITF increased the expression of these genes (Fig. 6E-6F).These results suggest that MITF directly represses the expression of these SASP genes, which in turn leads to resistance by suppressing senescence in resistant cells.
To determine whether O-GlcNAcylation of MITF regulates the expression of these SASP genes, we reconstituted MITF-depleted cells with WT MITF and S49A mutants, followed by qPCR analyses.
Intriguingly, transcriptional inhibition of these SASP genes was restored only upon the reexpression of WT MITF, but not the S49A mutant, in resistant cells with endogenous MITF depletion (Fig. 6G-6H).Consistently, only ectopic expression of WT MITF was able to overcome senescence induced by MITF knockdown, as evidenced by reduced p21 expression and SA-β-Gal staining, whereas such an effect was not observed in cells expressing the S49A mutant (Fig. 6I-6J).These data collectively indicate that O-GlcNAcylation of MITF at S49 is essential for its activity in the regulation of senescence-associated secretome in palbociclib-resistant cells.
MITF-mediated pathway is activated in response to palbociclib and elevated in tumors from palbociclibresistant breast cancer patients We next conducted clinical studies to investigate whether or not elevated OGT-MITF pathway is seen in patients who are undergoing CDK4/6i treatment or have developed resistance to CDK4/6i.Given that increased MITF levels contribute to palbociclib resistance, it is possible that palbociclib treatment may result in increased expression of MITF as a compensatory survival mechanism.Indeed, both MITF mRNA and protein expression levels were increased upon palbociclib treatment in MCF7 cells (Fig. 7A).The similar pattern was also observed in cells in response to the treatment of another CDK4/6 inhibitor ribociclib (Supp Fig. 6A).We next employed an ER positive breast cancer patient-derived xenograft (PDX) model to evaluate the effects of prolonged palbociclib treatment.Of note, the administration of palbociclib treatment to PDX tumor-bearing mice also led to a substantial increase in the expression level of MITF compared to the group without receiving the drug (Supp Fig. 6B).Thus, both in vitro and in vivo studies indicate that palbociclib treatment can stimulate MITF expression.
These data encouraged us to test whether the MITF pathway is also activated in patients receiving palbociclib treatment.To this end, we analyzed the gene expression data of patient tumors obtained from the NeoPalAna clinical trial, which included estrogen receptor-positive breast cancer patients undergoing tumor biopsies before and after palbociclib treatment 44 .In line with the aforementioned ndings in the preclinical setting, palbociclib treatment signi cantly increased MITF mRNA expression 16-20 weeks after palbociclib administration (surgery stage) (Fig. 7B and Supp Fig. 6C).To further explore the role of MITF in the regulation of palbociclib resistance in breast cancer, we created a MITF signature geneset by overlapping proteins that interact with MITF (mass-spec analysis) and genes that are known to be associated with MITF function (PahtwayNet analysis) (Supp Fig. 6D).Strikingly, the MITF signature geneset was signi cantly enriched in patients after palbociclib treatment compared to pre-treatment (Fig. 7C).Consistently, the same signature geneset was also elevated in the MCF-7 PR cells compared with its sensitive counterpart MCF7 cells (Supp Fig. 6E).
To further validate whether increased MITF levels are a critical factor regulating palbociclib resistance in breast cancer, we examined the expression of MITF-mediated pathway in a group of PDX lines, which consisted of 7 palbociclib-sensitive samples and 7 palbociclib-resistant samples.Signi cantly, expression levels of MITF, OGT, and O-GlcNAc were increased in most palbociclib-resistant PDX lines compared to sensitive lines (Fig. 7D and Supp Fig. 6F).Of note, both the MITF expression level and the O-GlcNAc expression levels were increased in the same PDX tumors before (ID# WHIM20, sensitive) and after (ID# WHIM20AR, resistance) the development of resistance to palbociclib due to long-term treatment with palbociclib in vivo, and MITF was predominantly located in the nuclei in resistant tumors (Fig. 7E-7F and Supp Fig. 6G).Taken together, these data from clinical samples strongly demonstrate that palbociclib treatment activates the MITF-mediated pathway and elevated MITF-mediated pathway is upregulated in palbociclib-resistant tumors.
Given that MITF mRNA is increased in MCF-7 PR cells and palbociclib-resistant patients as compared to their sensitive counterparts, and inhibition of OGT did not affect MITF mRNA level (Supp Fig. 6H), we next investigated how MITF mRNA is regulated in resistant cells.After analyzing patient data from the NeoPalAna clinical trial, we found a dramatic increase in the CREB pathway in patients who received palbociclib treatment (Fig. 7G).Since CREB directly regulates the transcription of MITF 55,56 , we assumed that the CREB pathway may regulate MITF mRNA expression in resistant cells.Indeed, the CREB pathway was remarkably enriched in palbociclib-resistant cell lines (Fig. 7H-7I and Supp Fig. 6I).Additionally, palbociclib treatment resulted in a dose-dependent increase of p-CREB expression, and inhibition of CREB pathway by using its speci c inhibitor 666 − 15 dramatically decreased MITF expression and re-sensitized resistant cells to palbociclib (Fig. 7J-7L and Supp Fig. 6J-6K).Thus, our cell-based studies and clinical evidence indicate that the CREB-dependent pathway plays an important role in the regulation of MITFmediated palbociclib resistance in breast cancer cells.

DISCUSSION
In this study, we have discovered a novel OGT-MITF-mediated pathway that plays a crucial role in regulating MITF transcriptional activity and conferring resistance to CDK4/6 inhibitors in breast cancer.Speci cally, we found that OGT-mediated O-GlcNAcylation of MITF at the S49 site within its nuclear localization signal (NLS) has a dual impact: it enhances the interaction between MITF and importin α/β, while concurrently impeding its association with 14-3-3.This intricate interplay results in the nuclear localization of MITF and subsequent activation of its transcriptional activity.In the context of resistant cells, the heightened MITF pathway contributes to palbociclib resistance by suppressing senescence through both O-GlcNAcylation and CREB-dependent mechanism (Fig. 7M).Notably, our study furnishes compelling clinical substantiation of the signi cance of the MITF-mediated pathway in response to palbociclib treatment.We bolstered our ndings with evidence from PDX lines and patients' tumors, further underlining the clinical relevance of this pathway.This study not only unveils a novel mechanism governing resistance to CDK4/6i but also offers a potential avenue for the future treatment of resistant breast cancer patients.
The identi cation of a novel mechanism involving GlcNAcylation in regulating MITF activity holds signi cant implications.Prior research has demonstrated that MITF activity can be in uenced by SUMOylation at two lysine residues, K182 and K316, which is believed to contribute to distinct target speci city 20,57 .Additionally, the mTOR pathway-induced phosphorylation of MITF has been observed, leading to heightened interaction with the 14-3-3 protein and subsequent retention within the cytosol 58 .
Our study introduces a fresh perspective by highlighting the role of OGT-mediated O-GlcNAcylation in governing MITF activity, primarily through its facilitation of nuclear translocation.However, the intricate interplay and potential cross-talk between phosphorylation, SUMOylation, and O-GlcNAcylation remain largely unexplored.If such interactions do exist, understanding their physiological signi cance in the context of cancer becomes crucial.One plausible scenario is that O-GlcNAcylation of MITF might compete with mTOR-mediated phosphorylation, exerting a dual in uence on both cellular distribution and activity of MITF.Further investigations aimed at unraveling the coordination between O-GlcNAcylation and the mTOR pathway, particularly at the structural level, could offer insights into the mechanism governing the development of palbociclib resistance in breast cancer cells.Such studies hold the potential to shed light on intricate regulatory networks and provide novel avenues for therapeutic intervention.
Importin α functions as an adaptor protein, recognizing and binding to cargo proteins containing NLSs, while importin β acts as a mediator, facilitating the transport of the importin α/cargo-protein complex through the nuclear pore complex (NPC) and into the nucleus 59 .Our ndings underscore the role of OGTmediated O-GlcNAcylation in MITF's interactions, as it enhances binding to importin α isoforms α4, α7, and α8.In contrast, the MITF S49 mutant displays reduced a nity for these importin proteins.Notably, S49 emerges as a pivotal residue within MITF's initial NLS region, playing a critical role in regulating its nuclear localization.Our data suggest that importin α functions as a reader for O-GlcNAcylated NLSs of MITF, thereby exerting control over its nuclear translocation.Intriguingly, O-GlcNAcylation of MITF at S49 disrupts its interaction with the 14-3-3 protein while simultaneously bolstering its association with importin proteins, facilitating nuclear translocation.This phenomenon hints at the potential for O-GlcNAcylation to induce conformational changes in MITF's protein structure.
MITF serves as a critical mediator of senescence in melanocytes and myeloma cells, primarily by orchestrating processes like cell cycle regulation, DNA repair, oxidative stress response, and the production of SASP factors.This is achieved through its modulation of downstream elements including SERPINE1, IL-6, and CCL2 60 .Despite its substantial role in senescence, the regulation of MITF itself in this context remains poorly understood.Our ChIP-seq analysis has unveiled the direct binding of MITF to promoter regions of key SASP-related genes such as SERPINE1, IL-6, CSF1, and PTGER2, signifying its role in governing their expression.Notably, our study underscores the essential role of O-GlcNAc-modi ed MITF in the suppression of senescence, as it negatively regulates the expression of SASP genes (Fig. 6).
Given that palbociclib induces cell cycle arrest and senescence as a primary mechanism to impede cell growth, we postulate that O-GlcNAc-MITF assumes a crucial role in modulating palbociclib resistance through senescence in breast cancer patients undergoing CDK4/6i therapy.The signi cance of O-GlcNAc-MITF in contributing to the development of resistance to CDK4/6i through senescence warrants further validation through rigorous clinical investigations.By shedding light on the intricate interplay between O-GlcNAc-MITF and palbociclib response, these studies have the potential to enhance our understanding of resistance mechanisms and pave the way for more effective therapeutic strategies.
Our investigation involving clinical samples has revealed a noteworthy observation -the administration of palbociclib leads to an upregulation in the expression of MITF and its associated gene set in breast cancer patients (Fig. 7).This signi cant nding suggests that CDK4/6i treatment triggers the elevation of the MITF-mediated pathway.This heightened pathway may potentially serve as a survival mechanism employed by cancer cells in response to CDK4/6 inhibition.This notion aligns with recent research that highlights elevated MITF expression in basal breast cancer patients, wherein its abundance is linked to unfavorable prognosis 61 .Our clinical studies contribute crucial evidence to support the proposition that MITF-mediated pathways are pivotal in orchestrating resistance to CDK4/6i among breast cancer patients.A particularly compelling instance involves the signi cantly increased MITF expression in the resistant PDX line obtained from long-term treatment with CDK4/6i in vivo (Fig. 7E).This patient-speci c data is both exhilarating and promising.In light of these ndings, we have embarked on a comprehensive clinical study encompassing a substantial cohort of patients receiving CDK4/6i treatment.This ongoing endeavor is aimed at amassing more robust clinical evidence to fortify our discovery.The ultimate aspiration is to propel this revelation towards potential clinical applications, thereby advancing our understanding of resistant mechanisms and fostering novel avenues for therapeutic intervention.

COMPETING INTERESTS
The authors declare no competing interests AUTHOR CONTRIBUTIONS Y.Z.performed most of the experiments with assistance from S.Y., Y.K., C.M., M.J., Z.Q., and J.F. designed and analyzed mass spectrometry data.M.H., Y.Q., and M.S. designed and performed the high-throughput drug screening assay.S.L. and C.M. provided the clinical patient samples.W.Z. supervised the project, cowrote the manuscript, provided experimental advice, and obtained funding to support the project.

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