Application of AFF1 and PARP1-AFF1 signal channels

Abstract

The invention discloses an application of AFF1 as a target spot in preparation of antitumor drugs and an application of a PARP1-AFF1 signaling pathway as a target spot in preparation of antitumor drugs. The invention also discloses a medicine which comprises an inhibitor, and the inhibitor is at least one of the following (1)-(3): (1) a preparation for inhibiting or knocking down or silencing the expression of AFF1; (2) a preparation for inhibiting a PARP1-AFF1 signal channel; and (3) a preparation for inhibiting the generation of poly ADP ribosylation modification of AFF1. According to the application disclosed by the invention, an action model of the PARP1-AFF1 signal channel in DNA damage repair is constructed, so that the understanding of a regulation mechanism of PARP1 in a DDR process is widened, and a potential target spot is provided for cancer treatment of a targeted DNA damage repair channel.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of AFF1 or PARP1-AFF1 signaling pathway as a target in the preparation of anti-tumor drugs. Background Art

[0002] The genome within a cell, carrying genetic material, is constantly exposed to endogenous and exogenous stressors, which can cause DNA damage and compromise genomic integrity. To cope with these threats, cells have evolved a surveillance mechanism called the DNA Damage Response (DDR) to identify and repair damage and maintain genomic stability.

[0003] Due to their rapid proliferation and uncontrolled division, tumor cells exhibit characteristics such as increased DNA damage and genomic instability. Radiotherapy and chemotherapy are based on this characteristic of tumor cells, treating cancer by inducing DNA damage and disrupting its repair pathways. However, tumor cells continue to evolve during treatment, leading to dysregulation of the DNA damage response and repair pathways, resulting in resistance to radiotherapy and chemotherapy, and ultimately leading to treatment failure. Therefore, research on the DNA damage response mechanism not only helps to reveal the basic laws of maintaining genomic stability, an important life activity, but also has key guiding significance for the prevention and treatment of diseases such as tumors.

[0004] PARP1 is the first member of the PARP family to be identified, and the family now includes 18 different members. PARP1 is a highly conserved, multifunctional enzyme that is widely found in eukaryotes. Its structure has been thoroughly studied and mainly consists of three domains (Figure 1): an amino-terminal DNA-binding domain (DBD) composed of three zinc finger motifs, which is responsible for recognizing and binding DNA; a BRCT domain containing a central self-modification domain, which plays a key role in the self-modification process of PARP1; and a highly conserved carboxyl-terminal catalytic domain, which is the core site of catalytic activity. These three domains work together to enable PARP1 to respond to various types of DNA damage.

[0005] Transcription is a key component of gene expression regulation. Protein-encoding genes are transcribed by RNA polymerase II (Pol II), a process involving complex steps and regulatory mechanisms. Gene transcription is closely linked to the DNA damage response. On the one hand, gene transcription is believed to promote rapid repair of DNA damage in a transcriptionally coupled manner; on the other hand, transcription itself is also a key endogenous factor that causes DNA damage and disrupts genomic stability. Furthermore, when faced with DNA damage, gene transcription must be rapidly shut down to prevent the synthesis of erroneous transcripts. Once repair is complete, transcription must be rapidly restarted to restore cellular life processes. Although transcription undergoes extensive reprogramming following DNA damage, how it coordinates with the DNA damage response to promote repair is only beginning to be understood. Therefore, clarifying the molecular interaction network between transcriptional regulation and the DNA damage response is crucial for further elucidating the regulatory mechanisms by which cells maintain genomic stability. Summary of the Invention

[0006] The purpose of the present invention is to provide applications and drugs of AFF1 and PARP1-AFF1 signaling pathway to assist in tumor treatment.

[0007] To achieve the above objectives, the present invention provides the following technical solutions: The present invention discloses the use of AFF1 as a target in the preparation of anti-tumor drugs. The application is to reduce and / or reverse tumor drug resistance. Furthermore, the drug inhibits, knocks down, or silences AFF1 expression.

[0008] In one embodiment, the tumor is nasopharyngeal carcinoma. Further, the AFF1 comprises the amino acid sequence shown in SEQ NO 1 in the sequence listing.

[0009] The present invention also discloses the use of the PARP1-AFF1 signaling pathway as a target in the preparation of an oncology drug. The application is to reduce and / or reverse tumor drug resistance. Furthermore, the drug inhibits the PARP1-AFF1 signaling pathway or inhibits poly (ADP-ribosylation) modification of AFF1.

[0010] In one embodiment, the tumor is nasopharyngeal carcinoma.

[0011] The present invention also discloses a drug comprising an inhibitor, wherein the inhibitor is at least one of the following (1)-(3):

[0012] (1) An agent that inhibits, knocks down, or silences the expression of AFF1.

[0013] (2) Preparations that inhibit the PARP1-AFF1 signaling pathway.

[0014] (3) Preparations that inhibit the poly(ADP-ribosylation) modification of AFF1.

[0015] Due to the adoption of the above scheme, the present invention has the following beneficial effects: the present invention discloses the use of AFF1 and the PARP1-AFF1 signaling pathway as targets in the preparation of anti-tumor drugs, and constructs a model of the role of the PARP1-AFF1 signaling pathway in DNA damage repair (DDR), which not only broadens the understanding of the regulatory mechanism of PARP1 in the DDR process, but also provides a potential target for cancer treatment targeting the DNA damage repair pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A Control or AFF1 knockout HeLa cells were left untreated or treated with 0.25 mM H₂O₂ for 5 minutes and allowed to recover for the indicated times. Newly synthesized RNA was analyzed by 5-EU labeling for 20 minutes. Right panel: Relative fluorescence intensity of each cell was quantified and normalized to that of the mock-treated group (set to 100). The red line indicates the mean intensity.

[0017] Figure 1B : Heat map of normalized read counts of nascent RNA (TT-seq) within a 100 kb window downstream of TSS in control or AFF1 KD cells at different time points after H2O2 treatment.

[0018] Figure 1C Genome browser maps showing TT-seq density of representative genes in control or AFF1 knockdown HeLa cells, untreated or after H2O2 treatment.

[0019] Figure 1D-1E : Meta-graphs showing the average TT-seq signal levels of (D) genes between 10-25 kb, 25-50 kb, and 50-100 kb or (F, G) AFF1 target genes in control cells and AFF1 KD cells at different time points after H2O2 treatment.

[0020] Figure 1F : Box plots show the log2 fold change in TT-seq signal levels of AFF1 target genes in control and AFF1 KD cells after 2.5 h of H2O2 treatment compared with untreated conditions.

[0021] Figure 1G :Gene ontology analysis of 2311 AFF1 target genes showing impaired transcriptional recovery in AFF1 KD cells.

[0022] Figure 2A HeLa cells were transfected with AFF1-F and treated with DMSO or 2 mM H2O2 for 30 min, 100 μM MNNG for 30 min, or 60 Gy IR followed by 10 min recovery or 20 J / m 2After 10 min of recovery from UV treatment, Flag-IP and WB were imaged using the LI-COR Odyssey dual-color infrared laser imaging system.

[0023] Figure 2B AFF1-F protein affinity-purified from WCEs of HeLa cells untreated or treated with the indicated doses of IR was analyzed by Western blotting.

[0024] Figure 2C : Western blot analysis of the input and immunoprecipitated AFF1-F proteins from HeLa cell WCEs that were untreated or treated with 2 mM H2O2 for 30 min was performed using three different antibodies (pan-ADPr, poly-ADPr, and mono-ADPr).

[0025] Figure 2D HeLa cells expressing AFF1-F protein were left untreated or treated with 2 mM H2O2 for 30 minutes. WCEs were incubated with af1521-Strep protein on beads for PD detection. Input and PD proteins were analyzed by Western blotting.

[0026] Figure 2E Left: Western blotting analysis of WCEs from HeLa cells or HeLa cells stably expressing AFF1-F. Right: Western blotting analysis of affinity-purified AFF1-F protein from WCEs of HeLa cells stably expressing AFF1-F, left untreated or treated with 2 mM H2O2 for 30 minutes.

[0027] Figure 2F SECs complexes CycT1-F, ELL2-F, AFF4-F, and AF9-F were affinity purified from HeLa cell WCEs that were untreated or treated with 2 mM H2O2 for 30 min and analyzed by Western blotting.

[0028] Figure 3A-3C HeLa cells were untreated or pretreated with (A) DNA-PKi / ATMi, (B) AZD2281, or (C) SK575, or were left untreated or treated with 2 mM H2O2 for 30 min. Affinity-purified AFF1-F protein was analyzed by Western blotting.

[0029] Figure 3D-Figure 3E : Affinity-purified AFF1-F protein from WCEs of control or (D) PARP1 KD or (E) PARP2 KD cells was analyzed by Western blotting.

[0030] Figure 3FAFF1-Strep was transfected into PARP1 KO cells complemented with PARP1-Flag WT or the enzymatically active E988A mutant. Cells were left untreated or treated with 2 mM H2O2 for 30 minutes. Affinity-purified AFF1-Strep protein and input protein were analyzed by Western blotting.

[0031] Figure 3G AFF1 protein affinity purified and bound to beads interacts with PARP1, HPF1, and NAD + Incubate for in vitro ADP-ribosylation reaction. After reaction termination and washing, the obtained samples were analyzed by Western blotting.

[0032] Figure 4A AFF1 KD cells complemented with stably expressed AFF1-WT or M2 proteins were left untreated or treated with 0.25 mM H2O2 for 5 minutes, followed by 5-EU labeling after a two-hour recovery period. Right: Relative fluorescence intensity of each cell was quantified and normalized to that of the mock-treated group (set to 100). The red line represents the mean intensity. Statistical analysis was performed using a two-tailed unpaired t-test; ****p < 0.0001.

[0033] Figure 4B AFF1 KD cells were complemented with stably expressed AFF1-WT or M2 protein and left untreated or treated with 0.25 mM H2O2 for 5 minutes, followed by a 2-hour recovery period and analyzed by Western Blot (WB).

[0034] Figure 4C HeLa cells pretreated with DMSO or AZD2281 were left untreated or treated with 0.25 mM H₂O₂ for 5 minutes and allowed to recover for the indicated times. Newly synthesized RNA was analyzed as shown. Right: Relative fluorescence intensity of each cell was quantified and normalized to the mock group (set to 100). The red line represents the mean intensity. Statistical analysis was performed using a two-tailed unpaired t-test; ****p < 0.0001.

[0035] Figure 5A -C: Affinity-purified full-length or truncated AFF1-F proteins from WCEs of (A, B) HeLa or (C) HPF1 KD cells untreated or treated with 2 mM H2O2 for 30 min were analyzed by Western blotting.

[0036] Figure 5D : Immunofluorescence staining of cells expressing AFF1-F mutant was performed using anti-Flag antibody.

[0037] Figure 5EWestern blotting analysis of full-length or truncated AFF1-F proteins purified from HeLa cell WCEs untreated or treated with 2 mM H2O2 for 30 min.

[0038] Figure 5F AFF1-601-900-F protein affinity purified and bound to beads binds to PARP1, HPF1 and NAD + After incubation for in vitro ADP-ribosylation reaction, the fixed AFF1-601-900-F protein was left untreated or treated with hydroxylamine or PARG, and then subjected to Western blotting analysis.

[0039] Figure 6 : Sequence of the AFF1 aa 601-900 region, where the 16 identified PARylation sites are highlighted in red and the remaining serine residues in the sequence are highlighted in blue.

[0040] Figure 7A Control cells, AFF1 KD cells, or AFF1 KD cells complemented with stably expressing AFF1-WT or M2 were left untreated or treated with the indicated doses of H2O2, and then subjected to a clonogenic assay. Relative colony counts were normalized to the untreated colony count as 100%.

[0041] Figure 7B Clonogenicity assays were performed on control or AFF1 KD HeLa cells that were unexposed or exposed to different doses of IR. Relative colony counts were normalized to the colony count of each cell line that was not exposed to IR as 100%.

[0042] Figure 7C AFF1 KD cells stably expressing AFF1-WT or M2 protein were complemented and treated with 5 Gy of IR and analyzed by immunofluorescence staining with γ-H2AX antibody at the indicated time points.

[0043] Figure 7D : Western blot detection of AFF1 knockdown efficiency.

[0044] Figure 7E Control or AFF1 KD HeLa cells were exposed to 2 Gy IR and then collected at the indicated time points for WB analysis.

[0045] Figure 7F Immunofluorescence staining of control and AFF1 KD HeLa cells treated with 5 Gy IR and 12 hours of post-treatment recovery using a γ-H2AX antibody. Right: Quantification of the number of γ-H2AX foci per cell. Red lines indicate the mean for each group.

[0046] Figure 7GControl or AFF1 KD HeLa cells were left untreated or treated with 0.25 mM H2O2 for 30 minutes and analyzed by comet assay. Quantification of the comet tail moment for each cell is shown. The red line represents the mean for each group.

[0047] Figure 8A :Clonogenic assay was performed on CNE2 or CR cells exposed to different doses of IR.

[0048] Figure 8B :The PAR modification levels in whole-cell lysates of CNE2 and CR cells that were not exposed or exposed to different doses of IR were analyzed by Western blotting.

[0049] Figure 8C CNE2 cells and CR cells without or without AZD2281 pretreatment were stained with crystal violet one week after exposure to 10 Gy IR.

[0050] Figure 8D :The proteins expressed in WCEs of CNE2 and CR cells were analyzed by Western blotting.

[0051] Figure 8E :The intensities of the indicated proteins in CNE2 and CR cells were measured by mass spectrometry.

[0052] Figure 8F : Scatter plots showing protein levels in CNE2 and CR cells after label-free quantification.

[0053] Figure 8G :The qRT-PCR method was used to detect the expression level of AFF1 mRNA in CNE2 and CR cells.

[0054] Figure 8H AFF1-F protein was affinity-purified from WCEs of CNE2 or CR cells that were unexposed or exposed to 10 Gy IR and recovered for 5 min, and analyzed by Western blotting.

[0055] Figure 8I CR cells pretreated with DMSO or AZD2281 were treated with CHX, and then samples were collected for WB analysis at the indicated time points.

[0056] Figure 9A-9B CR cells pretreated with DMSO or AZD2281 (A) or AFF1 knockdown (B) were treated with 5 Gy IR and allowed to recover for the indicated times. Newly synthesized RNA was analyzed as shown. Right: Relative fluorescence intensity was calculated and normalized to the control group in each group, set to 100. The red line represents the mean intensity. Statistical analysis was performed using a two-tailed unpaired t-test; ****p < 0.0001.

[0057] Figure 9C:Clonogenic assay of CNE2 or CR cells treated with different IR doses and statistical analysis.

[0058] Figure 9D :The AFF1 knockdown efficiency in CR cells was detected by WB.

[0059] Figure 9E SA-β-Gal staining was performed on control or AFF1 KD CR cells 3 days after 6 Gy IR treatment. Right panel: Quantification of SA-β-Gal-positive cells.

[0060] Figure 9F Control or AFF1 KD CR cells were exposed to 2 Gy IR, and then samples were collected at the indicated time points for WB analysis.

[0061] Figure 9G-Figure 9I Tumors formed in nude mice injected subcutaneously with CR cells were left untreated or treated with IR and / or AZD2281. (G) Tumor growth and (H) tumor weight were measured, and (I) tumors were removed and photographed.

[0062] Figure 9J-9L Tumors formed in nude mice after subcutaneous injection of control cells or AFF1 KD CR cells and IR treatment. (J) Tumor growth and (K) tumor weight were measured. (L) Tumors were then removed and photographed. DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is described in further detail below with reference to the accompanying drawings and specific embodiments.

[0064] Definitions related to the present invention

[0065] AFF1, short for AF4 / FMR2 family member 1, is a protein with important biological functions. It belongs to the AF4 / FMR2 family and plays a crucial role in transcriptional regulation and cell proliferation.

[0066] PARP1: Poly (ADP-ribose) polymerase 1, is an integral part of the DNA damage response (DDR) and a guardian of genomic integrity. PARP1 participates in the repair of DNA damage by recruiting multiple damage repair factors.

[0067] Poly (ADP-ribosylation) modification (PARylation) is a dynamic and reversible post-translational modification of proteins, which is maintained and regulated by a network consisting of a "writer" (PARP), an "eraser" (ADP-ribose hydrolase), a "reader" (ADPR binding domain) and a donor. The "poly" modification can be highly heterogeneous, with different numbers of ADP-ribose groups and linear and branched structures.

[0068] Pol II, also known as RNA polymerase II, is composed of 12 subunits. RPB1, the largest subunit, contains a unique tandem heptad repeat sequence in its carboxy-terminal domain (CTD). The amino acid residues within this sequence are highly susceptible to post-translational modifications, with phosphorylation and glycosylation being common.

[0069] DNA damage: DNA damage refers to abnormal changes in the structure of DNA molecules, which can affect the normal function of genes and even lead to cell death, cancer, or genetic diseases. It is the result of the combined effects of endogenous and exogenous factors in organisms.

[0070] Truncation: A portion of a protein that is shorter than its normal length.

[0071] Gene transcription: It is a biological process of synthesizing RNA using DNA as a template. Under the catalysis of RNA polymerase, one chain of DNA is used as a template and the RNA molecule is synthesized according to the principle of complementary base pairing.

[0072] Transcription restart / transcription recovery: refers to the process in which RNA polymerase (such as RNA Pol II) is reactivated and continues to extend the RNA chain through a specific mechanism after encountering pause, stagnation or external interference (such as DNA damage, regulatory factor action) during gene transcription.

[0073] PARP1 KD cells: A cell model in which the expression of poly(ADP-ribose) polymerase 1 (PARP1) is reduced by genetic engineering techniques (such as RNA interference, shRNA, or CRISPR-Cas9).

[0074] IR treatment: Ionizing radiation treatment is an experimental or clinical intervention method that induces DNA damage in cells or organisms through high-energy radiation (such as X-rays, gamma rays or particle beams). It is widely used in cancer radiotherapy, DNA damage repair mechanism research and radiobiology experiments.

[0075] The experimental materials and methods involved in the present invention are as follows:

[0076] 1. Experimental Materials

[0077] Molecular cloning and plasmid amplification and transformation were performed using competent cells prepared from Stabl3 strains, and protein prokaryotic expression and purification were performed using competent cells prepared from DE3 strains.

[0078] HeLa cells and HEK293T cells were obtained from ATCC, and CNE2 and CR cells were obtained from Xiangya Hospital of Central South University.

[0079] 2. Experimental Methods

[0080] The experimental methods of the present invention include common biological experiments such as: cell recovery, culture, passaging and freezing, competent cell preparation, plasmid transformation, plasmid extraction, protein immunoblotting WB, SDS-PAGE electrophoresis, Dot blot, protein bacterial prokaryotic purification, chromatin-bound protein separation, whole cell lysis and immunoprecipitation, TMT labeling mass spectrometry sample preparation, in vitro ADP ribosylation reaction, TT-Seq, Micro-irradiation experiment, comet assay, cell rescue experiment (rescue experiment), clone formation experiment, etc.

[0081] The present invention discloses the use of AFF1 or PARP1-AFF1 signaling pathway as a target in the preparation of anti-tumor drugs. Anti-tumor drugs exert their effects by inducing DNA damage. The core mechanism of most traditional chemotherapy drugs and radiotherapy is to directly or indirectly cause DNA damage, and tumor cells are more sensitive to DNA damage due to their active proliferation. Chemotherapeutic drugs (such as alkylating agents, platinums) directly destroy DNA structure (cross-linking, breakage); radiotherapy produces DNA double-strand breaks through ionizing radiation. The drugs of the present invention inhibit DNA damage repair, target the DNA repair pathway, weaken the repair ability of tumor cells, and amplify the chemotherapy / radiotherapy effect. The following is further detailed through specific examples.

[0082] Example 1

[0083] This example discloses the use of AFF1 as a target in the preparation of anti-tumor drugs. Furthermore, this application is intended to reduce and / or reverse tumor drug resistance. Drugs inhibit, knock down, or silence AFF1 expression to achieve anti-tumor effects. Experimental studies have demonstrated that AFF1 deficiency inhibits transcriptional recovery and impairs cell survival and DNA repair, providing a potential target for cancer therapy targeting DNA damage repair pathways.

[0084] The following research and experiments are conducted to demonstrate the technical solution of this embodiment.

[0085] 1. Loss of AFF1 inhibits transcriptional recovery

[0086] The levels of newly synthesized RNA were measured by pulse labeling of nascent transcripts with 5-ethyluridine (EU) and then imaging analysis at different time points after H2O2 treatment ( Figure 1A ). Control and AFF1 KD cells showed a strong global transcriptional block immediately after exposure to H2O2. However, at subsequent time points, they recovered to varying degrees. In control cells, transcription levels were almost completely restored after 2.5 h of H2O2 treatment, but in AFF1 KD cells, transcription levels failed to recover ( Figure 1A ).

[0087] To verify the transcriptional recovery defect in AFF1 KD cells on a genome-wide scale, transient transcriptome sequencing (TT-seq) was performed to assess the nascent transcription at different times after H2O2 treatment. Compared with control cells, transcriptional recovery in AFF1 KD cells was significantly impaired ( Figure 1B ,C). Notably, this defect was independent of gene length ( Figure 1D ). Through the list of AFF1 target genes, it was found that these genes also showed impaired transcriptional recovery in AFF1 KD cells, as shown in the meta-graph and box plot analysis ( Figure 1E -F). The analysis found that genes whose defects were restored in AFF1 KD cells were enriched in pathways essential for cellular response to stress, including regulation of mitosis and cell cycle, DNA metabolic processes, and translation regulation ( Figure 1G These results reveal the importance of AFF1 in transcriptional recovery after DNA damage.

[0088] 2. AFF1 undergoes PARylation modification under DNA damage

[0089] Western blot analysis was performed on AFF1 immunoprecipitated from cells exposed to different genotoxic stress inducers. Under conditions known to activate PARP1, such as hydrogen peroxide (H2O2), methylnitrosoguanidine (MNNG), and ionizing radiation (IR), poly(ADP-ribose) (PAR) signals co-migrated with immunoprecipitated AFF1, indicating that PAR was covalently bound to AFF1 ( Figure 2A ). Experiments have shown that although ionizing radiation induces AFF1 poly (ADP-ribosylation) (PARylation) in a dose-dependent manner ( Figure 2B ), but ultraviolet irradiation failed to promote the PARylation of AFF1. In addition, after hydrogen peroxide treatment, the present invention used pan-ADPr and poly-ADPr antibodies to detect and confirmed that AFF1 was modified by poly-ADP-ribosylation rather than mono-ADP-ribosylation ( Figure 2C ).

[0090] To confirm these findings, reverse pull-down experiments were performed using af1521, a bait protein known to enrich for PARylated proteins. As expected, the pull-down of AFF1 by af1521 was significantly enhanced after hydrogen peroxide treatment (2D).

[0091] To exclude the possibility that PARylation of AFF1 is caused by its transient overexpression, the present invention constructed a stable cell line that endogenously expresses AFF1 with a 3×Flag tag. Using this cell line, it was confirmed that AFF1 undergoes PARylation after hydrogen peroxide treatment ( Figure 2E The present invention also constructed plasmids of other SEC complex proteins with tags and detected their modifications under PARP1 activation. Among these subunits, only AFF1 and the previously discovered CycT1 were modified with PARylation after hydrogen peroxide treatment, which eliminated the interference of other subunits in the detection of PARylation modification on AFF1 ( Figure 2F ).

[0092] 3. PARylation modification of AFF1 depends on PARP1

[0093] The present invention further studies the role of PARP1 in mediating the PARylation of AFF1. Experiments show that chemical inhibition of ATM or DNA-PK activity does not change the PARylation of AFF1 after hydrogen peroxide treatment. Figure 3A ), pretreatment of cells with the selective inhibitor of PARP1 / 2, AZD2281, or the highly efficient and specific protease-targeted chimera (PROTAC) degrader, SK575, completely abolished hydrogen peroxide-induced PARylation of AFF1 ( Figure 3B , C). Therefore, in order to further determine whether PARP1 or PARP2 mediates the modification of AFF1, the present invention constructed PARP1 and PARP2 knockdown cell lines respectively. Experiments have shown that the loss of PARP1 but not PARP2 inhibits the PARylation of AFF1 ( Figure 3D In PARP1 knockout (KO) cells, complementation with wild-type PARP1 restored hydrogen peroxide-induced PARylation of AFF1, whereas complementation with the PARP1 enzyme-active mutant E988A failed to restore the PARylation (Figure F), indicating that PARylation of AFF1 requires PARP1 and can only be mediated when PARP1 is activated.

[0094] Finally, the direct PARylation of AFF1 by PARP1 was further verified by in vitro ADP-ribosylation reaction. + Under the catalysis of recombinant HPF1 and PARP1 proteins, AFF1 can effectively undergo PARylation modification ( Figure 3G ), which more directly illustrates the modification effect of PARP1 on AFF1. Taken together, these data confirm that activated PARP1 induces PARylation modification of AFF1 under DNA damage.

[0095] PARylation-mediated AFF1 stabilization promotes transcriptional recovery

[0096] To determine whether the impaired transcriptional recovery in AFF1-deficient cells is functionally related to its PARylation, rescue experiments were performed using stable cells expressing AFF1 WT or M2 in the absence of endogenous AFF1. Consistent with this, H2O2-induced transcriptional repression was largely restored in AFF1-WT cells but not in AFF1-M2 cells, as measured by 5-EU pulse labeling ( Figure 4A AFF1-M2 cells also exhibited reduced levels of Pol II pSer2 compared to AFF1-WT cells, reflecting the complementation of this transcriptional recovery defect in the M2 modification mutant cells ( Figure 4B Furthermore, inhibition of PARP1 by AZD2281 impaired transcriptional recovery after H2O2 treatment ( Figure 4C Together, these findings suggest that PARylation-mediated AFF1 stabilization is essential for efficient transcriptional recovery under conditions of DNA damage.

[0097] 5. The 601-900 region of AFF1 is rich in ADP-ribosylation sites

[0098] To facilitate the identification of ADP-ribosylation sites on AFF1, the present invention aims to identify the minimal region of AFF1 where PARylation occurs. After modification detection and analysis of a series of AFF1 truncations, the present invention found that the 601-900 region of AFF1 is both necessary and sufficient for hydrogen peroxide-induced PARylation of AFF1 ( Figure 5A -B). Like the full-length protein, H2O2-induced PARylation of AFF1601-900 also occurs in an HPF1-dependent manner ( Figure 5CWithin this region, three truncations were further constructed while retaining the nuclear localization sequence (NLS), namely AFF1Δ601-700, Δ701-811, and Δ835-900, and the correct distribution of these truncated AFF1 in the cell nucleus was confirmed ( Figure 5D The results showed that although the extent of PARylation of these truncations was somewhat reduced compared to the full-length protein after H2O2 treatment, their PARylation was not completely abolished compared to AFF1Δ601-900 ( Figure 5E Therefore, the present invention focuses on the AFF1601-900 region, the amino acid sequence of which is shown in Table 1 below and in the sequence listing SEQ NO 1.

[0099] Table 1. AFF1 601-900 region sequence

[0100] 601-650 GTKQPKKPVK ASARAGSRTS LQGEREPGLL PYGSRDQTSK DKPKVKTKGR 651-700 PRAAASNEPK PAVPPSSEKK KHKSSLPAPS KALSGPEPAK DNVEDRTPEH 701-750 FALVPLTESQ GQQHSGSGSG TSGCRQAVVV QEDSRKDRLPLPLRDTKLLS 751-800 PLRDTPPPQS LMVKITLDLL SRIPQPPGKG SRQRKAEDKQ PPAGKKHSSE 801-850 KRSSDSSSKL AKKRKGEAER DCDNKKIRLE KEIKSQSSSS SSSHKESSKT 851-900 KPSRPSSQSS KKEMLPPPPV SSSSQKPAKP ALKRSRREAD TCGQDPPKSA

[0101] In addition, using purified AFF1601-900 as a substrate, the present invention also observed in vitro modification experiments that the fragment can be mediated by poly ADP ribosylation modification ( Figure 5F Notably, after adding HPF1 to the reaction, the poly(ADP-ribose) residues at amino acids 601-900 of AFF1 were resistant to hydroxylamine treatment, similar to the properties of the poly(ADP-ribose) residues on the full-length AFF1 protein in cells. Both cellular and in vitro studies have demonstrated that the 601-900 region of AFF1 is rich in ADP-ribosylation sites. Based on the above demonstration, the AFF1 of the present invention can be either the full-length AFF1 protein or a truncated version containing amino acids 601-900.

[0102] 6. Loss of AFF1 impairs cell survival and DNA repair

[0103] 1. Construction of AFF1-M1 and AFF1-M2 mutants

[0104] A mutant named AFF1-M1 was generated by replacing the residues identified by mass spectrometry at the ADP-ribosylation site on AFF1 with alanine ( Figure 6 , the amino acid sites marked in red were mutated to alanine). The transiently expressed AFF1-M1 mutant did show reduced PARylation modification under H2O2 induction, but it was not significantly inhibited. Given the difficulty in fully identifying the modification sites and the previous discovery that the main modification sites are located on serine residues, and considering that PARP1 modifies other serine residues as new targets after mutation of these major modification sites, we further mutated the remaining serine residues in the 601-900 region of AFF1-M1 to alanine, generating the M2 mutant ( Figure 6, the sites marked in red and blue were mutated to alanine).

[0105] 2. Clone formation experiment

[0106] Through clone formation experiments, the cell viability was analyzed after treatment with different concentration gradients of hydrogen peroxide and different doses of ionizing radiation in control and AFF1 knockdown cells. The results showed that compared with control cells, AFF1-deficient cells were more sensitive to H2O2 or IR treatment ( Figure 7A -B).

[0107] After IR treatment, the intracellular γ-H2AX level of AFF1 KD cells was higher than that of control cells after 12 hours of recovery ( Figure 7D -F), indicating that DNA damage in cells cannot be quickly and effectively repaired after knocking down AFF1. Consistent with this result, the comet assay further confirmed that the DNA tails in AFF1 KD cells were longer and the degree of DNA damage was higher ( Figure 7G ).

[0108] The above is the knockdown of the entire AFF1 protein. In order to exclude the experimental results deviation caused by factors such as the disruption of its binding to other proteins after AFF1 knockdown, such as the disruption of the assembly of the AFF1 constitutive SEC complex by knockdown of AFF1, the present invention uses the AFF1-M2 modified mutant that has been constructed and verified to have the ability to assemble the SEC complex to construct cells that complement the wild type and M2 mutant after AFF1 knockdown. After treatment with the same experimental conditions, it was found that the weakened cell survival and DNA repair ability of AFF1 KD cells can be restored by complementing the expression of AFF1-WT ( Figure 7C This result fully demonstrates that AFF1 PARylation modification and protein stability are the key to cells' ability to quickly repair damage and survive DNA damage.

[0109] Example 2

[0110] This example discloses the use of the PARP1-AFF1 signaling pathway as a target in the preparation of an oncology drug. Furthermore, this application is intended to reduce and / or reverse tumor drug resistance. The drug inhibits the PARP1-AFF1 signaling pathway or inhibits poly(ADP-ribosylation) modification of AFF1 to achieve anti-tumor effects. Building on the research conducted in Example 1, the following research and experiments are conducted to demonstrate the technical solutions of this example.

[0111] 7. Ionizing radiation-resistant tumor cells accumulate AFF1 in a PARP1-dependent manner

[0112] Radiotherapy induces cytotoxicity by producing various types of DNA damage and is the mainstay of treatment for malignant tumors. However, the emergence of radioresistance in tumor cells is a major challenge that limits the clinical effectiveness of radiotherapy. Using a pair of nasopharyngeal carcinoma cell lines, CNE2, and its derivative, CNE2-IR resistant (hereafter referred to as CR), it was found that CR cells had a higher level of IR resistance than CNE2 cells ( Figure 8A ) and PARP1 activity ( Figure 8B At the same time, the present invention also proves that the increase in PARP1 activity in CR cells is not due to changes in PARP1 or PARG protein levels ( Figure 8D More importantly, it appears to play a key role in IR therapy resistance, as inhibition of PARP1 activity sensitizes CR cells to IR therapy ( Figure 8C ).

[0113] The present invention conducted proteomic analysis on CNE2 and CR cells and found that the abundance of AFF1 in CR cells increased ( Figure 8E ,F), western blot analysis further confirmed this conclusion ( Figure 8D The increase in AFF1 levels in CR cells is regulated at the posttranscriptional level, as we detected that AFF1 mRNA levels remained unchanged between CR and CNE2 cells ( Figure 8G In addition, due to the increased PARP1 activity, the PARylation of AFF1 in CR cells was significantly increased under IR treatment conditions ( Figure 8H Consistent with the findings in HeLa, AFF1 was relatively stable in CR cells but became highly unstable after PARP1 inhibition ( Figure 8I This demonstrates that elevated PARP1 activity in CR cells leads to the accumulation of AFF1 through poly(ADP-ribosylation)-mediated stabilization.

[0114] Together, these data suggest that enhanced PARP1 activity in CR cells promotes the stabilization of AFF1, thereby contributing to the resistance of CR cells to IR.

[0115] 8. PARP1-AFF1 signaling pathway contributes to radiation resistance

[0116] The present invention further investigated how dysregulation of the PARP1-AFF1 signaling pathway in CR cells promotes IR resistance. Similar to HeLa cells, blocking PARP1 activity or depleting AFF1 in CR cells severely impaired transcriptional recovery after IR treatment ( Figure 9A , B). AFF1 KD CR cells also showed reduced cell viability after exposure to IR ( Figure 9C , D). In addition, compared with control CR cells, AFF1-deficient CR cells induced senescence after IR treatment, and the accumulation of γ-H2AX levels was also higher ( Figure 9E , F).

[0117] The present invention further verified the above research results in vivo using a xenograft mouse model. Although inhibiting PARP1 activity alone did not significantly inhibit the growth of tumors formed by CR cells, it enhanced the radiotherapy sensitivity of CR cells and inhibited the growth of xenograft tumors ( Figure 9G -I). Notably, AFF1 deletion did not significantly attenuate CR cell proliferation under basal conditions, which is consistent with the view that AFF1-containing SECs constitute only a small fraction of the total SEC population and play a more important role under specific conditions, such as viral transactivation or cellular stress response. Knockdown of AFF1 combined with radiotherapy enhanced tumor suppression ( Figure 9J These results indicate that interfering with the PARP1-AFF1 signaling pathway can restore the sensitivity of radiotherapy-resistant tumors to radiotherapy.

[0118] Through research on radioresistant nasopharyngeal carcinoma cells, the present invention discovered that active PARP1 and stabilized AFF1 proteins contribute to their enhanced radioresistance, revealing the toxicity of the PARP1-AFF1 signaling pathway. Disrupting this signaling pathway can increase the sensitivity of cancer cells and tumors to radiotherapy. This not only broadens our understanding of the regulatory mechanisms of PARP1 in the DDR process but also provides a potential target for cancer therapy targeting the DNA damage repair pathway, thus providing a basis for the application of the present invention.

[0119] Example 3

[0120] This embodiment discloses a drug, including an inhibitor, wherein the inhibitor is at least one of the following (1)-(3):

[0121] (1) An agent that inhibits, knocks down, or silences the expression of AFF1.

[0122] (2) Agents that inhibit the PARP1-AFF1 signaling pathway: These may be substances that inhibit the activity of proteins associated with the PARP1-AFF1 signaling pathway or substances that inhibit the synthesis of proteins associated with the PARP1-AFF1 signaling pathway.

[0123] (3) Preparations that inhibit the poly(ADP-ribosylation) modification of AFF1.

[0124] In addition to the inhibitor, the drug of the present invention also includes other anti-tumor drugs (such as chemotherapy, targeted therapy or immunotherapy drugs), thereby improving drug resistance and synergistically enhancing the therapeutic effect.

[0125] The above description is only a preferred embodiment of the present invention. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention.

Claims

1. Application of AFF1 as a target in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The application is to reduce and / or reverse tumor drug resistance.

3. The use according to claim 1, characterized in that: The drug inhibits or knocks down or silences the expression of AFF1.

4. The use according to claim 1, characterized in that: The tumor is nasopharyngeal carcinoma.

5. The use according to any one of claims 1 to 4, characterized in that: The AFF1 includes the amino acid sequence shown in the sequence list SEQ NO1.

6. Application of PARP1-AFF1 signaling pathway as a target in the preparation of tumor drugs.

7. The use according to claim 6, characterized in that: The application is to reduce and / or reverse tumor drug resistance.

8. The use according to claim 6, characterized in that: The drug inhibits the PARP1-AFF1 signaling pathway or inhibits the poly ADP-ribosylation modification of AFF1.

9. The use according to claim 6, characterized in that: The tumor is nasopharyngeal carcinoma.

10. A drug, characterized in that Including an inhibitor, the inhibitor is at least one of the following (1)-(3): (1) Preparations that inhibit, knock down, or silence the expression of AFF1; (2) agents that inhibit the PARP1-AFF1 signaling pathway; (3) Preparations that inhibit the poly(ADP-ribosylation) modification of AFF1.

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