Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

Lei, Hu; Xu, Han-Zhang; Shan, Hui-Zhuang; Liu, Meng; Lu, Ying; Fang, Zhi-Xiao; Jin, Jin; Jing, Bo; Xiao, Xin-Hua; Gao, Shen-Meng; Gao, Feng-Hou; Xia, Li; Yang, Li; Liu, Li-Gen; Wang, Wei-Wei; Liu, Chuan-Xu; Tong, Yin; Wu, Yun-Zhao; Zheng, Jun-Ke; Chen, Guo-Qiang; Zhou, Li; Wu, Ying-Li

doi:10.1038/s41467-020-20259-0

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Published: 04 January 2021

Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

Hu Lei ORCID: orcid.org/0000-0003-3288-5205¹^na1,
Han-Zhang Xu¹^na1,
Hui-Zhuang Shan¹^na1,
Meng Liu¹,
Ying Lu¹,
Zhi-Xiao Fang¹,
Jin Jin¹,
Bo Jing¹,
Xin-Hua Xiao¹,
Shen-Meng Gao²,
Feng-Hou Gao³,
Li Xia¹,
Li Yang¹,
Li-Gen Liu¹,
Wei-Wei Wang¹,
Chuan-Xu Liu⁴,
Yin Tong⁵,
Yun-Zhao Wu¹,
Jun-Ke Zheng¹,
Guo-Qiang Chen ORCID: orcid.org/0000-0003-4936-2363⁶,
Li Zhou ORCID: orcid.org/0000-0002-9965-7533⁷ &
…
Ying-Li Wu ORCID: orcid.org/0000-0001-7909-8757¹

Nature Communications volume 12, Article number: 51 (2021) Cite this article

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Abstract

Identifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABL^T315I-induced CML in mice with the reduction of Lin⁻Sca1⁺c-Kit⁺ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

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Introduction

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy characterized by the t(9;22)(q34;q11) balanced reciprocal translocation of the Philadelphia (Ph) chromosome, which leads to the generation of BCR-ABL oncogenic fusion gene that encodes the chimeric BCR-ABL protein with constitutive kinase activity^1,2. The introduction of imatinib (IM) in 2001, a tyrosine kinase inhibitor (TKI) that targets BCR-ABL, revolutionized the prognosis of CML³. However, TKI resistance, including BCR-ABL-dependent and -independent resistance, is a major problem in IM-based CML treatment. The BCR-ABL-dependent mechanism is mainly mediated through the mutation of the ABL kinase domain, BCR-ABL overexpression, or MDR1 upregulation^4,5,6,7. To overcome IM resistance, the second generation of TKIs, such as dasatinib and nilotinib, has been developed⁸. For the “gatekeeper” mutation T315I, which confers resistance to all first- and second-generation TKIs, the third-generation TKI ponatinib was developed⁹. Nevertheless, the toxicity of ponatinib limits its use in some patients¹⁰. On the other hand, the underlying mechanisms of BCR-ABL-independent TKI resistance are still not well understood. It has been reported that the leukemia stem cells (LSCs) in CML are insensitive to TKI in a BCR-ABL-independent manner, thereby leading to relapse and minimal residual disease (MRD)¹¹. Additionally, the aberrant activation of the PI3K and RAS/MAPK signaling pathways also contributes to BCR-ABL-independent TKI resistance^12,13,14,15. Hence, identifying promising drug targets that overcome TKI resistance via both mechanisms is urgently required to provide new possibilities for CML treatment.

To date, ~100 kinds of deubiquitinating enzymes (DUBs) have been identified¹⁶. DUBs remove ubiquitin conjugates from their substrates, thereby altering their stabilities, localizations, or activities¹⁷. Accumulating evidence shows that DUBs are promising targets for cancer treatment, including hematopoietic malignancies. For instance, USP10 is involved in the pathogenesis of FLT3-ITD-positive leukemia¹⁸; targeting USP1 and USP7 is effective in multiple myeloma cells^19,20; and USP37 can stabilize PLZF/RARA in acute promyelocytic leukemia²¹. Although USP9X has been demonstrated to be involved in the survival of CML, how DUBs are related to CML pathogenesis is largely unexplored^22,23.

Ubiquitin-specific peptidase 47 (USP47) is a member of the USP subfamily of DUBs²⁴. Similar to other USPs, USP47 regulates cellular activities by removing ubiquitin conjugates from diverse substrates and, thereby, altering their stabilities, localizations, or activities. Specifically, USP47 deubiquitinates and stabilizes MAPK²⁵, DNA polymerase β (Polβ)²⁶, E-cadherin²⁷, β-catenin²⁸, SNAIL²⁹, YAP³⁰, β-Trcp³¹, and katanin-p60³². Hence, USP47 is involved in cell proliferation³³, cell survival³¹, DNA damage repair²⁶, NLRP3 inflammasome activation³⁴, and epithelial-mesenchymal transition²⁹. USP47 plays an important role in cancers such as gastric cancer, medulloblastoma, and colorectal cancer^35,36,37; however, its role in CML remains unexplored.

In this study, we reveal the critical role of USP47 in the pathogenesis of CML. Specifically, we demonstrate that USP47 is highly expressed in primary CML cells and promotes cell proliferation, while Usp47 knockout significantly prolongs the survival of BCR-ABL and BCR-ABL^T315I-induced CML mice by reducing leukemia stem/progenitor cells. We further demonstrate that USP47 facilitates DNA damage repair by regulating a novel substrate, Y-box binding protein 1 (YB-1). Moreover, we find that P22077, a USP47 inhibitor³⁸, substantially eliminates TKI-sensitive cells, TKI-resistant cells, leukemia stem/progenitor cells, and MRD in CML. We propose that USP47 is a promising target to overcome TKI resistance in CML treatment.

Results

BCR-ABL regulates USP47 through RAS/ERK and STAT5 pathway in CML

To screen the potential DUBs involved in the pathogenesis of CML, we compared the expression of DUBs in primary CML cells (chronic phase, n = 5) and normal bone marrow (BM) CD34⁺ cells (n = 3). Compared with normal BM CD34⁺ cells, some of the DUBs including USP47, USP9X, USP14, OTUB2, etc. are expressed at higher levels in CML cells, with USP47 being the most upregulated one (Fig. 1a). Consistent with this result, the USP47 protein level is significantly upregulated in primary CML cells at different stages of disease progression (Fig. 1b). Next, we determined whether USP47 is regulated by BCR-ABL. Compared with the cells in the control group, the expression of USP47 at the mRNA (Supplementary Fig. 1a) and protein (Fig. 1c) levels are significantly upregulated in BCR-ABL-transfected myeloid progenitor 32D cells (32D^BCR−ABL). In contrast, BCR-ABL knockdown or IM treatment attenuates the expression of USP47 at the mRNA and protein levels (Fig. 1d–f). Furthermore, the expression of USP47 is attenuated after RAS or ERK inhibition and STAT5 silence (Fig. 1g, h and Supplementary Fig. 1b, c). These results suggest that BCR-ABL-induced upregulation of USP47 is mediated by the activation of RAS/ERK and STAT5 signaling pathways.

USP47 knockdown inhibits proliferation of CML cells

To investigate the role of USP47 in CML, USP47 was silenced in IM-sensitive K562, IM-resistant K562R, and KBM5^T315I cells. As depicted in Fig. 2a, the proliferation of these three cell lines is compromised. Moreover, the numbers of sub-G1 and G2/M phase cells increase after USP47 knockdown (Fig. 2b). The caspase-3 activity is also increased in USP47-knockdown cells (Fig. 2c), indicating the activation of apoptosis in CML cells. Interestingly, USP47 knockdown in human normal BM CD34⁺ cells does not inhibit cell viability or colony formation ability (Fig. 2d–f). To investigate the function of USP47 in vivo, USP47-silenced and control K562 cells were subcutaneously injected into nude mice. Compared with the control group, knockdown of USP47 remarkably reduces the tumor volumes (Supplementary Fig. 2a–c). Immunohistochemical staining shows that the percentages of PCNA-positive cells are significantly reduced, whereas the percentages of γH₂AX (a well-recognized DNA damage marker) positive cells are markedly increased compared with the control group (Supplementary Fig. 2d). These data suggest that USP47 is involved in promoting CML cell proliferation both in vitro and in vivo.

**Fig. 2: Knockdown of *USP47* inhibits the proliferation of CML cells.**

Usp47 knockout suppresses BCR-ABL-induced CML in mice

To further explore the role of USP47 in CML, the Usp47 knockout mouse model (Usp47^−/−) was established (Fig. 3a). Compared with Usp47^+/+ mice, the numbers of total BM cells, total white blood cells, red blood cells, granulocytes, lymphocytes, and platelets are comparable with those in Usp47^−/− mice (Fig. 3b and Supplementary Fig. 3a). The hematopoietic systems of Usp47^−/− and Usp47^+/+ mice are also similar (Supplementary Fig. 3b, c). Moreover, the percentages of the bone marrow Lin⁻Sca1⁺c-kit⁺ (LSK) cells, which are the target cells for BCR-ABL, are also comparable between Usp47^+/+ and Usp47^−/− mice (Fig. 3c). However, the mice received BCR-ABL-transformed Usp47^−/− BM cells survived significantly longer than those receiving BCR-ABL-transformed Usp47^+/+ cells (Fig. 3d). Moreover, the size and weight of the spleens in Usp47^+/+ mice are significantly increased than those of the Usp47^−/− mice (Fig. 3e). Flow cytometry analysis showed that the percentage of GFP⁺Gr-1⁺ cells in BM is significantly lower in the Usp47^−/− group compared with that in the Usp47^+/+ group (Fig. 3f, upper panel). Consistently, fewer CML leukemia cells were detected in the PB of Usp47^−/− group (Fig. 3f, lower panel). Also, the liver and spleen tissues were more seriously damaged in the Usp47^+/+ group than in the Usp47^−/− group (Fig. 3g). In addition, substantial infiltration of CML leukemia cells (GFP-positive cells) was detected in the liver and spleen of the Usp47^+/+ mice (Fig. 3h). Furthermore, the number of CML leukemia stem/progenitor cells (GFP⁺ LSK cells) in the Usp47^−/− group is significantly decreased compared with the Usp47^+/+ group (Fig. 3i). These data demonstrate that USP47 plays a vital role in the pathogenesis of BCR-ABL-induced CML.

**Fig. 3: *Usp47* knockout suppresses the development of BCR-ABL-induced CML in mice.**

Silencing Usp47 inhibits BCR-ABL^T315I-induced CML and proliferation of KBM^T315I in a xenograft mouse model

Next, we examined the role of Usp47 in a BCR-ABL^T315I-driven CML mouse model. Consistent with the findings above, Usp47 depletion substantially prolongs the survival of CML mice compared with the Usp47^+/+ group (Fig. 4a). Compared with Usp47^+/+ mice, the spleen size and weight in Usp47^−/− mice are significantly decreased (Fig. 4b, c). Besides, there is a remarkable decrease in the number of CML leukemia cells in the PB and BM of the Usp47^−/− mice (Fig. 4d, e). Flow cytometry analysis shows that the number of GFP⁺Gr-1⁺ cells is comparable between the Usp47^+/+ and Usp47^−/− mice at 14 days after CML model establishment, while the number of GFP⁺Gr-1⁺ cells decreases in Usp47^−/− mice as time goes by (Supplementary Fig. 4a). The liver and spleen tissues are severely damaged in the Usp47^+/+ group compared with the Usp47^−/− mice (Fig. 4f). Additionally, Usp47 knockout does not affect the homing efficacy of CML LSK cells (Supplementary Fig. 4b). However, the number of CML GFP⁺ LSK cells in the Usp47^−/− group is significantly lower than that in the Usp47^+/+ group (Supplementary Fig. 4c). To further validate this finding in human CML cells, we inoculated the USP47-knockdown KBM5^T315I (ShUSP47-2) and the control KBM5^T315I (Ctrl shRNA) cells into B-NDG mice. Compared with the control group, the mice in the ShUSP47-2 group show more prolonged survival with smaller spleen size and weight (Fig. 4g, h). Moreover, leukemic infiltration in the livers of the control mice is more obvious than that of the ShUSP47-2 mice (Fig. 4i). Also, the hematoxylin and eosin (H&E) staining results show that the extent of leukemic infiltration in the liver and spleen in the ShUSP47-2 group is lower than the control group (Fig. 4j). These observations are further confirmed by CD45 antibody staining (Fig. 4k). These data further reveal the vital role of USP47 in the pathogenesis of CML with T315I mutation.

**Fig. 4: Silence of Usp47 inhibits BCR-ABL^T315I-induced CML and inhibits proliferation of KBM^T315I in a xenograft model.**

USP47 interacts with and stabilizes YB-1 protein

We explored the mechanism through which USP47 functions in CML. Although it has been reported that MAPK or β-catenin interacts with USP47 in Drosophila or tumors^25,28, USP47 knockdown does not affect the expression of MAPK or β-catenin in CML cells (Supplementary Fig. 5a). We then immunoprecipitated, separated, and analyzed the USP47-interacting proteins using mass spectrometry (Fig. 5a). It was identified that YB-1 can interact with USP47 in K562 and primary CML cells (Fig. 5b). To map the interaction domain of YB-1 with USP47, truncated forms of YB-1 (GFP tag) were constructed and co-transfected with USP47 (Flag tag) into HEK293T cells. The results show that USP47 interacts with the C-terminal domain (CTD) domain of YB-1 (Fig. 5c). We further examined if USP47 regulates the ubiquitination of YB-1. As shown in Fig. 5d, USP47 overexpression significantly inhibits ubiquitination of full-length YB-1 and YB-1-S3, but not that of the truncated YB-1-S2, which cannot bind USP47. Consistent with the deubiquitinating effect of USP47 on YB-1, less ubiquitinated YB-1 is observed in primary CML cells than that in normal BM cells (Fig. 5e). Interestingly, the interaction between YB-1 and USP47 is not affected by the ubiquitination status of YB-1, as the mutation of lysine residues (Lys137, −164, and −170) abrogates the ubiquitination of YB-1, but not its interaction with USP47 (Fig. 5f, g). We then investigated whether USP47 influences the stability of YB-1 and found that transient transfection of USP47 prolonged the half-life of YB-1 in K562 cells (Fig. 5h). In contrast, USP47 knockdown or P22077 treatment reduces the half-life of YB-1 (Fig. 5i and Supplementary Fig. 5b). The half-life of YB-1 is also reduced in Usp47 knockout MEF cells (Fig. 5j). The USP47 knockdown-induced downregulation of the YB-1 protein can be rescued by the pre-treatment of MG132, a proteasome inhibitor (Fig. 5k). Meanwhile, knockdown of USP47 has no effect on YB-1 or POLB at mRNA level and YB-1 mRNA expression is similar in normal and CML cells (Supplementary Fig. 5c, d), indicating the increased YB-1 protein levels in primary CML cells mainly attributes to the post-transcriptional regulation of YB-1. Moreover, YB-1 expression is restored in Usp47^−/− MEFs after the reintroduction of Usp47 (Fig. 5l). These findings suggest that YB-1 serves as a novel substrate for USP47.

**Fig. 5: USP47 interacts with YB-1 and protects it from proteasomal degradation.**

YB-1 contributes to USP47-mediated DNA damage repair in CML cells

We examined whether YB-1, a well-known multifunctional transcription factor and an oncoprotein in cancers^39,40,41,42, contributes to the proliferation suppression of CML cells induced by USP47 inhibition. As expected, YB-1 knockdown significantly inhibits the proliferation of K562, K562R, and KBM5^T315I cells (Fig. 6a). In agreement with previous studies that USP47 is involved in the DNA damage repair pathway^26,43, USP47 knockdown significantly increases γH₂AX and ATR phosphorylation (Ser428) in CML cells (Fig. 6b). Furthermore, Usp47 knockout significantly enhances the expression of γH₂AX in GFP⁺ BM cells from BCR-ABL^T315I-induced CML mice (Fig. 6c). These results were further confirmed by γH₂AX foci and the number of AP sites (DNA damage quantification analysis) (Fig. 6d, e). Time-course analysis of USP47 knockdown-induced DNA damage response shows that, with the decrease of USP47, γH₂AX increases, followed by the cleavage of PARP1, indicating that DNA damage response is not a secondary effect of cells already committed to apoptosis (Fig. 6f and Supplementary Fig. 6a). Consistent with previous findings that YB-1 regulates PCNA, epidermal growth factor (EGF), and DNA topoisomerase II α (TOPO IIα)^44,45,46, YB-1 knockdown reduces the mRNA expression of PCNA and TOPO IIα (Fig. 6g and Supplementary Fig. 6b), indicating YB-1 is involved in DNA damage response in CML cells. The role of YB-1 in DNA damage repair was further validated as we found that YB-1 knockdown induces the increase of ATR phosphorylation (Ser428), PAR, and γH₂AX in CML cells (Fig. 6h and Supplementary Fig. 6c). YB-1 knockdown also causes γH₂AX foci in CML cells (Fig. 6d and Supplementary Fig. 6d). γH₂AX clearance is also significantly slower in irradiation-induced YB-1 knockdown cells than in control cells (Fig. 6i). To assess the contribution of YB-1 and Polβ to USP47-mediated DNA damage repair in CML cells, we overexpressed USP47 in YB-1 and/or POLB knockdown K562 and KBM5^T315I cells. As shown in Supplementary Fig. 6e, knockdown of POLB cannot increase γH₂AX expression. USP47 overexpression in YB-1 and/or Polβ depletion cells cannot abrogate γH₂AX expression, indicating that YB-1 contributes more to USP47-mediated DNA damage repair than Polβ in CML cells (Supplementary Fig. 6f). As expected, overexpressing YB-1 attenuates DNA damage response induced by USP47 knockdown, as evidenced by the decrease of γH₂AX expression and the number of AP sites (Fig. 6j, k). Also, YB-1 overexpression in Usp47^−/− MEFs attenuates the upregulation of γH₂AX (Fig. 6l). Additionally, the utilization of AZD6738 (50 nM), an ATR pathway inhibitor, partially inhibits USP47 knockdown-induced suppression of cell viability in CML cells (Supplementary Fig. 6g). As DNA damage repair plays an essential role in the maintenance or generation of CML stem/progenitor cells^47,48, we hypothesized that DNA damage induced by USP47 knockdown might be vital to CML stem/progenitor cells. In agreement with our hypothesis, USP47 knockdown significantly decreases the colony formation ability of primary CML CD34⁺ cells (Fig. 6m). The number of γH₂AX foci also increases after USP47 knockdown in primary CD34⁺ cells (Fig. 6n). The results indicate that YB-1 is involved in USP47-mediated DNA damage repair in CML cells.

P22077 exerts remarkable toxicity on CML cells and CML LSCs in vitro and in vivo

P22077, a dual inhibitor of USP7/USP47, was used to assess the function of USP47 in CML cell lines and primary CML cells. As depicted in Fig. 7a, P22077 exhibits low toxicity to mononuclear cells from normal PB, while in contrast, P22077 inhibits the proliferation of both IM-sensitive and IM-resistant CML cell lines and primary CML cells in a dose-dependent manner (Fig. 7b–d). Notably, P22077 shows evident cytotoxicity to BM mononuclear cells derived from CML patients resistant to second-generation TKIs (Fig. 7e). In both primary IM-sensitive and IM-resistant CML cells, P22077 treatment decreases YB-1 but increases the expression of γH₂AX and the cleavage of caspase-3 and PARP1, indicating the activation of apoptosis (Fig. 7f). In addition, USP47 is positively correlated with the expression of YB-1 in primary CML cells, and CML cells with higher levels of USP47 are more sensitive to P22077 treatment than the cells with lower levels of USP47 (Supplementary Fig. 7a). We then evaluated the effect of P22077 on KBM5^T315I cells using a B-NDG mouse CML model. The B-NDG mice injected with KBM5^T315I cells by tail vein were divided into four groups and treated with the vehicle, IM, P22077, and P22077 plus imatinib, respectively. P22077 significantly prolongs the survival of CML mice, while P22077 combined with IM has no significant enhancement effect on P22077 (Fig. 7g). More importantly, using CML patient-derived xenograft model established from a TKI-resistant CML patient, we found that P22077 treatment could significantly prolong the survival of mice (Supplementary Fig. 7b) and reduce the percentages of CD34⁺CD38⁻ cells in the secondary bone marrow transplantation (Fig. 7h, I). Given the important role of USP47 in CML stem/progenitor cells, we investigated the effect of P22077 on primary CD34⁺ CML cells. P22077 exerts no cytotoxicity on normal CD34⁺ cells, but significantly inhibits the colony-forming activity of CD34⁺ cells from de novo and IM-resistant CML patients (Fig. 7j). Moreover, P22077 significantly decreases the number of CML stem/progenitor cells in a BCR-ABL-induced CML mouse model (Fig. 7k). P22077 treatment significantly eliminates the number of CML GFP⁺LSKs in the MRD mouse model (Fig. 7l). These results indicate that P22077 exerts cytotoxicity markedly against CML cells, including CML stem/progenitor cells.

**Fig. 7: P22077 shows remarkable toxicity on CML cells and CML leukemia stem/progenitor cells in vitro and in vivo.**

Discussion

TKI resistance and CML leukemia stem cells remain the major challenges in the treatment of BCR-ABL⁺ CML. In this study, we demonstrate that USP47 plays a critical role in the proliferation of TKI-sensitive, TKI-resistant, and CML stem/progenitor cells both in vitro and in vivo. As a substrate of USP47, YB-1 contributes to USP47-mediated DNA damage repair in CML cells. Hence, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate CML stem/progenitor cells.

The role of USP47 in cancer is cancer type-dependent. It can function as an oncoprotein in gastric cancer⁴⁹, colorectal^29,30, ovarian cancer⁵⁰, and a tumor suppressor in medulloblastoma³⁶. Our study reveals that USP47 is essential in the pathogenesis of CML. We show that knockout of Usp47 significantly prevents BCR-ABL or BCR-ABL^T315I-induced leukemogenesis and reduces the number of GFP⁺LSK cells in CML mice, indicating that Usp47 is critical in the development of BCR-ABL-induced CML. Moreover, targeting USP47 is effective in CML cells regardless of the presence of BCR-ABL with or without kinase domain mutation. CML cells derived from all analyzed CML patients (irrelevant to BCR-ABL mutation or TKI resistance) exhibit similar sensitivity to USP47 inhibition-induced cytotoxicity. Furthermore, USP47 inhibition subdues the proliferation of K562, K562R (without BCR-ABL mutation and insensitive to IM), and KBM5^T315I (with BCR-ABL T315I mutation and insensitive to IM) CML cell lines both in vitro and in vivo. Therefore, our study suggests that USP47 is a promising therapeutic target in CML treatment, especially for CML patients with TKI resistance.

Consistent with previous reports^26,43, we found that USP47 is involved in the DNA damage repair in CML cells. We further demonstrated that YB-1, a novel substrate of USP47, contributes to USP47-mediated DNA damage repair. In agreement with our findings, it has been reported that YB-1 is associated with cancer progression and drug resistance in multiple cancers by stimulating cell proliferation and promoting replicative immortality, genomic instability, and metastasis^{39,40,41,42,51,52,53}. Several studies reported that YB-1 is also involved in base excision and mismatch repair pathways by interacting with multiple DNA repair proteins, such as DNA ligase IIIα⁵⁴, APE1⁵⁵, MSH2⁵⁶, and PCNA⁵⁷. In CML, BCR-ABL compromises the fidelity of nucleotide excision repair and homologous recombination repair, thus promoting survival and resulting in the genomic instability of CML cells, especially LSCs^11,58,59,60. Therefore, it is reasonable that in CML cells, BCR-ABL induces DNA damage, while USP47 and YB-1 are required to repair DNA damage to ensure cell survival.

Another important finding is that P22077 is effective in TKI-sensitive and TKI-resistant CML cells, regardless of the presence of T315I mutation. Interestingly, in addition to inhibiting the activity of USP47, P22077 considerably reduces the protein level of USP47 in CML cells. P22077 may promote the degradation of USP47 by inhibiting the auto-deubiquitinating process of USP47²⁸, which is supported by the finding that MG132 reverses P22077-induced USP47 degradation (Supplementary Fig. 7c). It has been reported that P22077 also inhibits the activity of USP7 and USP10. To identify the specific USPs targeted by P22077 in CML cells, we measured the expression of substrates of USP7 and USP10, including p53, a substrate of both USP7 and USP10^61,62, and SIRT6, a substrate of USP10⁶³. We found that P22077 increases p53 expression (but not phosphorylated p53, ser15) in primary CML cells but does not affect SIRT6 expression. However, both p53 and SIRT6 expressions are not increased by P22077 treatment in KBM5^T315I cells (Fig. 7f). Also, USP10 or USP7 knockdown does not affect the expression of YB-1 (Supplementary Fig. 7d). Moreover, P22077 still strongly inhibits the viability of USP7 or USP10 stably knockdown K562 cells (Supplementary Fig. 7e). Therefore, we deem that USP47 is the main effector for P22077 to combat CML. In addition, it should be noted that, mice treated with P22077 (30 mg/kg) for 2 weeks did not show health problems such as weight loss (Supplementary Fig. 7f), which was consistent with the previous reports⁶⁴. These data indicate P22077 is a promising candidate compound for the treatment of TKI-sensitive and TKI-resistant CML. Nevertheless, further investigations are still needed to develop USP47-specific inhibitors for CML treatment.

Based on the fact that TKI reduces the expression of USP47 in CML cells and overexpression of BCR-ABL upregulates the expression of USP47 in 32D cells, we demonstrate that BCR-ABL upregulates the expression of USP47. However, BCR-ABL may not be the only regulator of USP47. First, the activation of RAS/ERK, which is independent of BCR-ABL, can regulate the expression of USP47^65,66. Second, USP47 is highly expressed in several types of BCR-ABL-negative cell lines, including colorectal cancer cells and gastric cancer cells^30,49. Therefore, USP47 may be regulated through both BCR-ABL-dependent and -independent mechanisms.

In summary, we demonstrate that USP47 is a target for CML treatment and targeting USP47 is a promising strategy for overcoming TKI resistance and eradicating leukemia stem/progenitor cells in CML.

Methods

Reagents and antibodies

The p-Bcr (WB, 1:1000, #3901, Lot 1), c-Abl (1:1000, #2862, Lot 13), p-CrkL (1:1000, #3181, Lot 7), CrkL (1:1000, #3182, Lot 5), p-ERK (1:1000, #9101, Lot 26), ERK (1:1000, #9102, Lot 25), p-P38 MAPK (1:1000, #9215, Lot 19), p-JNK MAPK (1:1000, #9251, Lot 10), p-AKT (1:1000, #4060, Lot 30), caspase-3 (1:1000, #9661, Lot 45), γH₂AX (1:1000, #9718, Lot 1), p-p53 (1:1000, #9284), p-ATR (1:1000, #2853, Lot 9), p-ATM (1:1000, #4526, Lot 14), β-catenin (1:1000, #9587, Lot 2) and PAR (1:1000, #83732) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The USP47 (1:500, sc-100633, Lot J1314), STAT5 (1:500, sc-74442), USP14 (1:500, sc-100630, Lot J3013), Ubiquitin (1:500, sc-8017, Lot H0409), GFP (1:500, sc-9996, Lot J0813), PARP1 (1:500, sc-56197), PCNA (1:500, sc-53407), p53 (1:500, sc-126) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for Polβ (1:1000, #18003-1-AP, Lot 00009472), YB-1 (1:1000, #20339-1-AP, Lot 00046294), Flag tag (1:1000, #66008⁻2-Ig), USP10 (1:1000, #19374-1-AP, Lot 00015015), TOPOIIα (1:1000, #20233-1-AP), SIRT6 (1:1000, #13572-1-AP, Lot 00004562), β-actin (1:1000, #HRP-66009) were purchased from Proteintech Group, Inc. (Wuhan, CHN). The USP7 (1:1000, #A300-033A) antibody was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA). The USP16 (1:1000, GTX16439, Lot 39631) antibody was purchased from GeneTex Inc. (Irvine, CA, USA). The above antibodies were used in western blot. Lineage Antibody Cocktail (1:200, #88-7772-72), sca-1 (1:200, #25-5981-82), c-kit (1:200, #17-1171-82), CD34 (1:200, #11-0349-42), CD38 (1:200, #12-0389-42) and CD45 (1:200, #17-0459-42) for FACS analysis were purchased from ebioscience Inc. (San Diego, CA, USA). Gr-1 antibody (1:200, #130-119-794) was purchased from Miltenyi Biotec (Auburn, CA, USA). CD45 antibody (1:1000, #GB14038) for IHC was purchased from ServiceBio (Wuhan, CHN). Flag-bead (M20018) was purchased from Abmart (Shanghai, CHN). EasySep Release Human CD45 Positive Selection Kit for Humanized Mice was purchased from STEMCELL (100-0107, Vancouver, BC, Canada). Imatinib (S2475), P22077 (S7133), and MG132 (S2619) were purchased from Selleck Chemicals (Houston, TX, USA). AZD6738 (T3338) was purchased from Target Molecule Corp. (Shanghai, CHN). Pan-RAS-IN-1 (HY-101295) was purchased from MedChemExpress LLC. (Monmouth Junction, NJ, USA). The Cell Cycle Detection Kit (340242) was purchased from BD Biosciences (San Jose, CA, USA). The Caspase-3 Activity Assay Kit (C1115) was purchased from Beyotime Biotechnology (Jiangsu, CHN).

Cell culture

The murine IL-3-dependent myeloid cell line 32D obtained from ATCC (CRL-11346) was maintained in RPMI-1640 (21875109; Gibco. Grand Island, New York, USA) supplemented with 10% fetal bovine serum (FBS) (900-108; Gemini Bio-Products Inc. Sacramento, USA) and 10% WEHI-3B conditional medium containing IL-3; their counterparts transformed with p210^BCR−ABL were maintained in RPMI-1640 (11875; Gibco) supplemented with 10% FBS. The IM-sensitive K562 cells obtained from ATCC (CRL-243) and IM-resistant cell lines K562R cells obtained from ATCC (CRL-3344), respectively, were cultured in RPMI-1640 (11875; Gibco) supplemented with 10% FBS. K562R cells were cultured in 1 μM IM to maintain their drug-resistant status. KBM5^T315I cells⁶⁷ were kindly provided by Jingxuan Pan in Sun Yat-sen University and were cultured in IMDM (12440061; Gibco) supplemented with 10% heat-inactivated FBS, penicillin (50 U/ml)/ streptomycin (50 μg/ml) (E607011; Sangon Biotech Co, Shanghai, CHN). All cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂. HEK293T cells were obtained from ATCC (CRL-3216) and cultured in DMEM (11971025, Gibco) with 10% FBS. All cell stocks were routinely tested for mycoplasma contamination and showed negative.

Primary cells

BM samples were obtained from healthy donors and patients with CML (from 2011 to 2019) at different clinical stages or at drug resistance admitted to Shanghai Jiao Tong University School of Medicine affiliated Ruijin Hospital, Xinhua Hospital, Shanghai First People’s Hospital, Shanghai 9th People’s Hospital and Shanghai Tongren Hospital. The details of patients were provided in Supplementary Table 1. Mononuclear cells were isolated by Ficoll-Paque Plus density gradient media (GE Life Sciences, Chicago, IL, USA). Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. All manipulations were approved by the Medical Ethic Committee of Shanghai Jiao Tong University School of Medicine.

RNA interference

ShRNA oligonucleotides targeting USP47, BCR-ABL, YB-1, STAT5, USP7, and USP10 were designed and synthesized (Supplementary Table 2). For gene silencing, USP47, BCR-ABL, YB-1, STAT5, POLB, USP7, and USP10-specific ShRNA and control ShRNA were cloned into the pSIREN-RetroQ vector. The retrovirus supernatant was packaged in HEK293T cells by cotransfecting with pSIREN-RetroQ, VSVG and gag-pol. The viral supernatant was collected after transfection for 48 h. The cells were treated with viral supernatant and polybrene (4 μg/ml) for 48 h.

Lentiviral transduction in CML cells

Lentiviruses were produced by transient transfection in HEK293T cells using pLVX-ZsGreen-IRES-YB-1 or pLVX-ZsGreen-IRES-USP47 plasmid, envelope plasmid pMD2.G, and packaging plasmid psPAX2. CML cells were infected twice by spinoculation (1500 × g, 60 min, room temperature) with virus-containing supernatants.

Cell cycle analysis

USP47-specific knockdown and control cells were collected separately. Then, the cells were rinsed with cold PBS and fixed in 75% ethanol at −20 °C for at least 24 h. Subsequently, the cells were rinsed again with cold PBS for two times, incubated with RNAase (10 μg/mL) at 37 °C for 30 min and stained with propidium iodide (PI; 50 μg/mL). Flow cytometry (BD FACS Calibur) was used to examine the percentages of cells at different cell cycle stages.

Immunoprecipitation and immunoblot analysis

Whole-cell extracts were prepared with lysis buffer (P0013; Beyotime, Jiangsu, CHN), then they were incubated with the appropriate antibody overnight at 4 °C. Protein A&G beads (sc-2003; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added, and the incubation was continued for 4 h at 4 °C. Beads were washed three times with PBS buffer. The bound proteins were then separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and probed with the appropriate antibodies. The image was visualized using a chemiluminescence imaging analyzer (GE, ImageQuant LAS 4000).

Mass spectrometry analysis

Immunoprecipitation samples were subjected to SDS-PAGE and visualized with colloidal Coomassie blue. Two obvious bands were cut out and digested by sequencing-grade modified trypsin (Promega). The tryptic peptides were analyzed by liquid chromatography with tandem mass spectrometry ((LC-MS/MS)) (liquid chromatography coupled with mass spectrometry). The MS experiments were performed on an LTQ orbitrap “XL” mass spectrometer assembled by an Easy-nLC 1000 via an Easy Spray (Thermo Fisher Scientific). The peptide mixture was separated in a 50-cm-column (inner diameter = 0.075 mm) packed with C18 2-mm Reversed Phase resins (PepMap RSLC) using 300-min linear gradient elution, from 95% solvent A (0.1% formic acid and 2% acetonitrile within 98% water) to 35% solvent B (0.1% formic acid and 2% water within 98% acetonitrile) at a flow rate of 200 nl/min. All MS/MS spectra were searched against the uniprot human database by Mascot Searching engine (Matrix Science, London, UK). Finally, the validated peptides and protein identification were exported by Scaffold (Proteome Software Inc., Portland, OR) using the Scaffold Local FDR algorithm.

Quantitative real-time PCR (RT-PCR)

Total RNA was extracted using Trizol reagent (Invitrogen Corp. Waltham, MA, USA), and cDNA was prepared using a Reverse Transcriptase kit (Takara Bio Inc. Shiga, Japan). SYBR Master Mix (Roche Diagnostics Corporation, Indianapolis, IN, USA) and the Applied Biosystems Step One PlusTM detection system (ABI 7900) were used for real-time quantitative PCR. The DUBs primer sequences (Supplementary Table 3) and specific primer sequences (Supplementary Table 4) were used to analyze gene expression.

CML mouse model

Usp47 knockout (Usp47^−/−) mice (C57BL/6 J background) were obtained from MMRRC (Mutant Mouse Resource & Research Centers, USA). To generate the Usp47^−/− mouse with BALB/c background, we backcrossed these mice to the BALB/c mice for over 10 generations. All subsequent Usp47-deficient (−/−) or wild-type (WT, + / + ) mice used in this study were generated from mating with littermates. The Usp47 genotype was determined by PCR analysis. The primers used were the following: β-Geo (580 bp) 5′-CAA ATG GCG ATT ACC GTT GA−3′, 5′-TGC CCA GTC ATA GCC GAA TA-3′; Usp47 (907 bp) 5′-CTT CAC CTG TTC AAA TCC TCC G-3′, 5′-GTT CCT TTC TGT TCA TAC CCG ATG-3′. The retroviral plasmid MIGR1 carrying the p210^BCR−ABL or T315I mutation p210^BCR−ABL was prepared by transient transfection with the Ecopack construct in HEK293T cells. BM cells from 5-fluorouracil-treated (200 mg/kg) Usp47^+/+ or Usp47^−/− BALB/c donor mice (8-week) were transduced twice with BCR-ABL retrovirus in the presence of interleukin-3 (IL-3), interleukin-6 (IL6) and stem cell factor (SCF). Wild-type recipient mice were subjected to 7 Gy γ-irradiation and followed by injection with infected BM cells (1 × 10⁶ per mouse) via tail vein injection. After transplantation, the incidence of CML in the recipient mice was observed and recorded.

The mice were bred in an equipped animal facility with the temperature at 20–25 °C and humidity at 30–70%, with a 12 h light–dark cycle and ad libitum access to regular chow diet and water. All animal procedures were approved by the committee for the humane treatment of animals at Shanghai Jiao Tong University School of Medicine. The study was compliant with all of the relevant ethical regulations regarding animal research.

B-NDG mouse model

Six-week-old NOD-SCID IL-2 receptor gamma (B-NDG) null female mice were obtained from Jiangsu Biocytogen Co., Ltd. (Nantong, CHN). KBM5^T315I cells (4 × 10⁶) transduced with control ShRNA or USP47 ShRNA were injected into the tail vein of mice (n = 8, each). The survival of mice from the tail vein model was analyzed and is shown with a Kaplan-Meier survival plot. The spleen from the sacrificed mice was measured to determine whether the mice had splenic enlargement. For drug administration experiments, KBM5^T315I cells (2 × 10⁶) were transplanted via tail vein injection into 6-week-old B-NDG mice. Between 15 and 30 days, the mice were treated through tail vein injection of vehicle (10% DMSO + 10% BASF-ELP + 10% 1,2-Propanediol + 70% physiological saline) (n = 6), P22077 (dissolved in the vehicle) (intraperitoneally, 30 mg/kg, n = 6), IM (intraperitoneally, 50 mg/kg, n = 6) or P22077 + IM (intraperitoneally, n = 6). The survival time of the mice was determined. Primary CML-R cells (5 × 10⁶) were transplanted via tail vein injection into 6-week-old B-NDG mice. Between 30 and 45 days, the mice were treated every day through tail vein injection of vehicle (n = 6), P22077 (dissolved in the vehicle) (intraperitoneally, 30 mg/kg, n = 6). For the secondary transplantation, human CD45 cells (2 × 10⁶) from BM were transplanted into two groups (n = 7) of B-NDG mice. The survival times of the mice were determined, and the percentages of CD34⁺CD38⁻ cells in human CD45 cells from BM were measured. The graphical account for FACS (BD FACS Calibur) sequential gating/sorting strategies was provided in Supplementary Fig. 8.

Immunohistochemical and H&E staining

The samples were fixed with 4% paraformaldehyde for 2 days, then dehydrated through a graded series of ethanol and embedded in paraffin. Sections were cut and stained with haematoxylin-eosin (H&E). For immunohistochemistry, the samples were incubated overnight at 4 °C with primary antibodies after antigen retrieval in citrate buffer. The samples were incubated for 30 min with biotinylated second antibody IgG and then for 20 min with Streptavidin-HRP peroxidase. The reaction products were visualized with diaminobenzidine (DAB)-H₂O₂ as a substrate for peroxidase. All sections were counterstained with hematoxylin. The image was visualized using a microscope (Olympus, CKX31).

Immunofluorescence staining

The cultured cells were fixed to a slide by a cytospin and fixed with 0.3% Triton X-100. After permeabilization with 100% cold methanol and blocking with 2% (w/v) bovine serum albumin, the cells were incubated γH₂AX antibody (1:100 dilution) overnight at 4 °C. Then, the slides were stained with the secondary antibody and DAPI. The fluorescence signal of the cells was detected by confocal microscopy (Nikon, A1R-si).

DNA damage quantification

Genomic DNA was isolated and purified using the QIAamp DNA Mini Kit (51304; QIAGEN, Hilden, Germany). The number of apurinic/apyrimidinic sites (AP sites) was determined using the DNA Damage Quantification Kit (DK02, DOJINDO Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction.

Cell viability assay

CML cell lines and primary CML cells were incubated with different concentrations of P22077 and/or IM for 48 h. Cell viability was assayed using a Cell Counting Kit-8 (CK04, DOJINDO Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Colony-forming assay

CD34⁺ cells from BM mononuclear cells of CML or normal healthy volunteers were obtained using human CD34⁺ selection cocktail (14756, STEMCELL, Vancouver, BC, Canada). CD34⁺ cells were infected with USP47 knockdown lentivirus for 24 h, and then the cells were seeded at 1000 cells per well of a 12-well plate. Different concentrations of P22077 and 1000 CD34⁺ cells were mixed with the stem cell medium (H4434, STEMCELL), and then continuously incubated at 37 °C in 5% humidified CO₂. After incubation for 14 days, the colonies were counted.

MRD mouse model

BCR-ABL retrovirus-infected mouse BM cells were transplanted into 6-week-old female wild-type mice, and the GFP⁺Gr-1⁺ cells were detected by FACS on day 21. The CML mice were treated with IM (50 mg/kg, intraperitoneally) for 40 days, and then the mice were randomly divided into two groups. One group was treated with P22077 (30 mg/kg, intraperitoneally), the other group was given vehicle as control. The effect of P22077 on GFP⁺LSKs in BM was examined by FACS after 12 days.

Mouse xenograft model

K562 control and USP47 stably knockdown cells (8 × 10⁶) were implanted subcutaneously into the right flanks of 6-week-old female Balb/c (nu/nu) mice (Slac Laboratory Animal Co., Ltd., China). Tumor sizes were measured using calipers, and tumor volumes were calculated using a standard formula (width² × length/2). Tumor cell proliferation and apoptosis were detected by hematoxylin and eosin (H&E) staining, PCNA immunohistochemical (IHC) staining, and TUNEL assays were performed.

Homing analysis

We transduced BM from Usp47^−/− and Usp47^+/+ mice (n = 3, each group, 8-week-old) with BCR-ABL retrovirus for 48 h. GFP⁺LSK cells in infected BM cells were measured by FACS. The infected BM cells (before transplantation) were then injected into lethally irradiated 7.5 Gy Usp47^+/+ mice. The BM cells were collected 18 h after injection (after transplantation). The GFP⁺LSK cells were monitored by FACS. The homing efficacy was calculated by the ratio of the percentage of GFP⁺LSK cells: [GFP⁺LSK] after transplantation 18 h/ [GFP⁺LSK] before transplantation.

Statistics and reproducibility

All statistical tests were performed using GraphPad Prism 6 (GraphPad Software Inc.). All data are presented as the mean ± sd. Student’s t-test was used to determine significant differences between two groups. One-way or two-way analysis of variance (ANOVA) was used to analyze the significant differences among multiple groups. For all statistical tests, p-values < 0.05 were considered to be statistically significant. The number of independent experiments and replicates (n) is indicated in the figure legends. At least three biologically independent replicates were performed for each experiment and similar results were obtained.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The data supporting the findings of this study are available within the article and its supplementary materials, which are provided as a Source Data file. In addition, some raw data was generated in the Core Facility of Basic Medical Sciences of our school. Derived data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported in part by grants from the National Key Research and Development Program of China (No. 2017YFA0505202), the National Basic Research Program of China (973 Program) (No. 2015CB910403), the National Natural Science Foundation of China (81570118, 81700475, 81570112, 81670139), CAMS Innovation Fund for Medical Sciences (CIFMS): 2019-I2M-5-051, Natural Science Foundation of Shanghai (19ZR1428700). We thank the excellent technical support from Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine.

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These authors contributed equally: Hu Lei, Han-Zhang Xu, Hui-Zhuang Shan.

Authors and Affiliations

Hongqiao International Institute of Medicine, Shanghai Tongren Hospital/Faculty of Basic Medicine, Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, 200025, Shanghai, China
Hu Lei, Han-Zhang Xu, Hui-Zhuang Shan, Meng Liu, Ying Lu, Zhi-Xiao Fang, Jin Jin, Bo Jing, Xin-Hua Xiao, Li Xia, Li Yang, Li-Gen Liu, Wei-Wei Wang, Yun-Zhao Wu, Jun-Ke Zheng & Ying-Li Wu
Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical University, 325000, Wenzhou, China
Shen-Meng Gao
Department of Oncology, Shanghai 9th People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhi Zao Ju Road, 200011, Shanghai, China
Feng-Hou Gao
Department of Hematology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 200092, Shanghai, China
Chuan-Xu Liu
Department of Hematology, Shanghai First People’s Hospital, Shanghai Jiao Tong University School of Medicine, 200081, Shanghai, China
Yin Tong
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education and Chinese Academy of Medical Sciences Research Unit (NO.2019RU043), Shanghai Jiao Tong University School of Medicine, 200025, Shanghai, China
Guo-Qiang Chen
Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, 200025, Shanghai, China
Li Zhou

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Hu Lei
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Han-Zhang Xu
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Hui-Zhuang Shan
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Meng Liu
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Ying Lu
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Zhi-Xiao Fang
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Jin Jin
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Bo Jing
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Xin-Hua Xiao
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Shen-Meng Gao
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Feng-Hou Gao
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Li Xia
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Li Yang
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Li-Gen Liu
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Wei-Wei Wang
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Chuan-Xu Liu
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Yin Tong
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Yun-Zhao Wu
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Jun-Ke Zheng
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Guo-Qiang Chen
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Li Zhou
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Ying-Li Wu
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Contributions

The experiments were conceived and designed by H.L., J.Z., L.Z., and Y.W. The plasmids were constructed by H.L., H.X., and B.J. H.L., H.S., X.X., and Y.L. performed animal experiments. The CML patient samples were collected by H.L., L.Z., S.G., F.G., H.X., Z.F., L.Y., L.L., Y.T., and C.L. M.L., J.J., Z.W., and W.W. did some experiments about P22077 and signal pathway. L. Xia provided mass spectrometry technical support. The paper was written and revised by H.L., G.C., and Y.W.

Corresponding authors

Correspondence to Guo-Qiang Chen, Li Zhou or Ying-Li Wu.

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Lei, H., Xu, HZ., Shan, HZ. et al. Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia. Nat Commun 12, 51 (2021). https://doi.org/10.1038/s41467-020-20259-0

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Received: 15 January 2017
Accepted: 23 November 2020
Published: 04 January 2021
DOI: https://doi.org/10.1038/s41467-020-20259-0

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