Preclinical testing of CT1113, a novel USP28 inhibitor, for the treatment of T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive and heterogeneous lymphoid malignancy with poor prognosis in adult patients. Aberrant activation of the NOTCH1 signalling pathway is involved in the pathogenesis of over 60% of T-ALL cases. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase known to regulate the stability of NOTCH1. Here, we report that genetic depletion of USP28 or using CT1113, a potent small molecule targeting USP28, can strongly destabilize NOTCH1 and inhibit the growth of T-ALL cells. Moreover, we show that USP28 also regulates the stability of sterol regulatory element binding protein 1 (SREBP1), which has been reported to mediate increased lipogenesis in tumour cells. As the most critical transcription factor involved in regulating lipogenesis, SREBP1 plays an important role in the metabolism of T-ALL. Therefore, USP28 may be a potential therapeutic target, and CT1113 may be a promising novel drug for T-ALL with or without mutant NOTCH1.

The deubiquitinase USP28 maintains the expression of the transcription factor MYCN and is essential in neuroblastoma cells.

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children. MYCN gene amplification is highly associated with poor prognosis in high-risk NB patients. In non-MYCN-amplified high-risk NB patients, the expression of c-MYC (MYCC) and its target genes is highly elevated. USP28 as a deubiquitinase is known to regulate the stability of MYCC. We show here USP28 also regulates the stability of MYCN. Genetic depletion or pharmacologic inhibition of the deubiquitinase strongly destabilizes MYCN and stops the growth of NB cells that overexpress MYCN. In addition, MYCC could be similarly destabilized in non-MYCN NB cells by compromising USP28 function. Our results strongly suggest USP28 as a therapeutic target for NB with or without MYCN amplification/overexpression.

Functional Phosphoproteomics in Cancer Chemoresistance Using CRISPR-Mediated Base Editors.

Selective inhibition of targeted protein kinases is an effective therapeutic approach for treatment of human malignancies, which interferes phosphorylation of cellular substrates. However, a drug-imposed selection creates pressures for tumor cells to acquire chemoresistance-conferring mutations or activating alternative pathways, which can bypass the inhibitory effects of kinase inhibitors. Thus, identifying downstream phospho-substrates conferring drug resistance is of great importance for developing poly-pharmacological and targeted therapies. To identify functional phosphorylation sites involved in 5-fluorouracil (5-FU) resistance during its treatment of colorectal cancer cells, CRISPR-mediated cytosine base editor (CBE) and adenine base editor (ABE) are utilized for functional screens by mutating phosphorylated amino acids with two libraries specifically targeting 7779 and 10 149 phosphorylation sites. Among the top enriched gRNAs-induced gain-of-function mutants, the target genes are involved in cell cycle and post-translational covalent modifications. Moreover, several substrates of RSK2 and PAK4 kinases are discovered as main effectors in responding to 5-FU chemotherapy, and combinational treatment of colorectal cancer cells with 5-FU and RSK2 inhibitor or PAK4 inhibitor can largely inhibit cell growth and enhance cell apoptosis through a RSK2/TP53BP1/γ-H2AX phosphorylation signaling axis. It is proposed that this screen approach can be used for functional phosphoproteomics in chemotherapy of various human diseases.

Ubiquitin-specific protease 28 deubiquitinates TCF7L2 to govern the action of the Wnt signaling pathway in hepatic carcinoma.

Overexpression of ubiquitin-specific protease 28 (USP28) is found in hepatic carcinoma. It is unclear whether the deubiquitinase plays a role in hepatocarcinogenesis. Deregulation of the Wnt signaling pathway is frequently associated with liver cancer. Transcription factor 7-like 2 (TCF7L2) is an important downstream transcription factor of the Wnt/ß-catenin signaling pathway, but the mechanisms by which TCF7L2 itself is regulated have not yet been revealed. Here, we report that USP28 promotes the activity of the Wnt signaling pathway through maintaining the stability of TCF7L2. We further show that FBXW7 is the E3 ubiquitin ligase for TCF7L2. By regulating the levels of TCF7L2, USP28 modulates the Wnt/ß-catenin signaling in liver cancer and USP28 depletion or inhibition by a small molecule inhibitor leads to a halt of growth in liver cancer cells. These results suggest that USP28 could be a potential therapeutic target for liver cancer.

Cdh1 Deficiency Sensitizes TNBC Cells to PARP Inhibitors.

Triple-negative breast cancer (TNBC) is a type of breast tumor that currently lacks options for targeted therapy. Tremendous effort has been made to identify treatment targets for TNBC. Here, we report that the expression level of anaphase promoting complex (APC) coactivator Cdh1 in TNBC is elevated compared to that in the adjacent healthy tissues, and high levels of Cdh1 expression are correlated with poor prognoses, suggesting that Cdh1 contributes to the progression of TNBC. Interfering with the function of Cdh1 can potentiate the cytotoxic effects of PARP inhibitors against BRCA-deficient and BRCA-proficient TNBC cells through inducing DNA damage, checkpoint activation, cell cycle arrest, and apoptosis. Further investigation reveals that Cdh1 promotes BRCA1 foci formation and prevents untangled DNA entering mitosis in response to PARP inhibition (PARPi) in TNBC cells. Collectively, these results suggest that APC/Cdh1 is a potential molecular target for PARPi-based therapies against TNBCs.

Engineering Novel CD19/CD22 Dual-Target CAR-T Cells for Improved Anti-Tumor Activity.

Despite high remission rates following chimeric antigen receptor T cell (CAR-T) cell therapy in B-cell acute lymphoblastic leukemia (B-ALL), relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may improve the CAR-T effect. The in vitro and in vivo leukemia model was established, and the anti-tumor effects of BiCAR-T, CD19 CAR-T, CD22 CAR-T, and LoopCAR6 cells were observed. We found that the BiCAR-T cells showed significant cytotoxicity in vitro and in vivo. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce the risk of antigen loss.

The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) lacks significant expression of the estrogen receptor, the progesterone receptor, and of human epidermal growth factor receptor. It is the most aggressive and malignant of all breast cancers, and for which, there are currently no effective targeted therapies. We have shown previously that the RecQ helicase family member RECQL5 is essential for the proliferation and survival of TNBC cells; however, the mechanism of its involvement in cell viability has not been shown. Here, we report that the expression of RecQ family helicases, including RECQL5, is regulated by the deubiquitinase USP28. We found using genetic depletion or a small molecule inhibitor that like RECQL5, USP28 is also essential for TNBC cells to proliferate in vitro and in vivo. Compromising the function of USP28 by shRNA knockdown or the inhibitor caused TNBC cells to arrest in S/G2 phases, concurrent with DNA-damage checkpoint activation. We further showed that the small molecule inhibitor of USP28 displayed anti-tumor activity against xenografts derived from TNBC cells. Our results suggest that USP28 could be a potential therapeutic target for triple negative breast cancer.

Resistance and recurrence of malignancies after CAR-T cell therapy.

The emergence of chimeric antigen receptor T (CAR-T) cell therapy has ushered a new era in cancer therapy, especially the treatment of hematological malignancies. However, resistance and recurrence still occur in some patients after CAR-T cell treatment. CAR-T cell inefficiency and tumor escape have emerged as the main challenges for the long-term disease control of B cell malignancies by this promising immunotherapy. In solid tumor treatment, CAR-T cells must also overcome many hurdles from the tumor or immune-suppressed tumor environment, which have become obstacles to the advancement of CAR-T therapy. Therefore, an understanding of the mechanisms underlying post-CAR treatment failure in patients is necessary. In this review, we characterize some mechanisms of resistance and recurrence after CAR-T cell therapy and correspondingly suggest reasonable treatment strategies.

Transcription-Replication Collisions and Chromosome Fragility.

Accurate replication of the entire genome is critical for cell division and propagation. Certain regions in the genome, such as fragile sites (common fragile sites, rare fragile sites, early replicating fragile sites), rDNA and telomeres, are intrinsically difficult to replicate, especially in the presence of replication stress caused by, for example, oncogene activation during tumor development. Therefore, these regions are particularly prone to deletions and chromosome rearrangements during tumorigenesis, rendering chromosome fragility. Although, the mechanism underlying their "difficult-to-replicate" nature and genomic instability is still not fully understood, accumulating evidence suggests transcription might be a major source of endogenous replication stress (RS) leading to chromosome fragility. Here, we provide an updated overview of how transcription affects chromosome fragility. Furthermore, we will use the well characterized common fragile sites (CFSs) as a model to discuss pathways involved in offsetting transcription-induced RS at these loci with a focus on the recently discovered atypical DNA synthesis repair pathway Mitotic DNA Synthesis (MiDAS).

RECQL5 plays an essential role in maintaining genome stability and viability of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a malignancy that currently lacks targeted therapies. The majority of TNBCs can be characterized as basal-like and has an expression profile enriched with genes involved in DNA damage repair and checkpoint response. Here, we report that TNBC cells are under replication stress and are constantly generating DNA double-strand breaks, which is not seen in non-TNBC cells. Consequently, we found that RECQL5, which encodes a RecQ family DNA helicase involved in many aspects of DNA metabolism including replication and repair, was essential for TNBC cells to survive and proliferate in vitro and in vivo. Compromising RECQL5 function in TNBC cells results in persistence of DNA damage, G2 arrest, and ultimately, cessation of proliferation. Our results suggest RECQL5 may be a potential therapeutic target for TNBC.

Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice.

Liver sinusoidal endothelial cells (LSECs) have great capacity for liver regeneration, and this capacity can easily switch to profibrotic phenotype, which is still poorly understood. In this study, we elucidated a potential target in LSECs for regenerative treatment that can bypass fibrosis during chronic liver injury. Proregenerative LSECs can be transformed to profibrotic phenotype after 4 weeks of carbon tetrachloride administration or 10 days of bile duct ligation. This phenotypic alternation of LSECs was mediated by extracellular regulated protein kinases 1 and 2 (Erk1/2)-Akt axis switch in LSECs during chronic liver injury; Erk1/2 was normally associated with maintenance of the LSEC proregenerative phenotype, inhibiting hepatic stellate cell (HSC) activation and promoting tissue repair by enhancing nitric oxide (NO)/reactive oxygen species (ROS) ratio and increasing expression of hepatic growth factor (HGF) and Wingless-type MMTV integration site family member 2 (Wnt2). Alternatively, Akt induced LSEC profibrotic phenotype, which mainly stimulated HSC activation and concomitant senescence by reducing NO/ROS ratio and decreasing HGF/Wnt2 expression. LSEC-targeted adenovirus or drug particle to promote Erk1/2 activity can alleviate liver fibrosis, accelerate fibrosis resolution, and enhance liver regeneration. This study demonstrated that the Erk1/2-Akt axis acted as a switch to regulate the proregenerative and profibrotic phenotypes of LSECs, and targeted therapy promoted liver regeneration while bypassing fibrosis, providing clues for a more effective treatment of liver diseases.

Disruption of peroxisome function leads to metabolic stress, mTOR inhibition, and lethality in liver cancer cells.

Peroxisome houses a large number of enzymes involved in lipid and phytochemical oxidation as well as synthesis of bile acid and other specialized lipids. Peroxisome resident enzymes are imported into the organelle via a conserved cargo transport system composed of many peroxins, protein factors essential for the biogenesis of peroxisome. Among the peroxins, PEX5 plays a transporter role, and PEX2, 10, and 12 are thought to form a complex that functions as an E3 ubiquitin ligase to help recycle PEX5 in an ubiquitin modification-dependent process. Previous studies have demonstrated the importance of peroxins in postnatal development especially the development of nerve systems. These studies also show that peroxins or the function of peroxisomes is dispensable for cellular viability. In contrast, however, we report here that PEX2 and other peroxins are essential for the viability of liver cancer cells, probably through altering metabolism and signaling pathways. Our results suggest that peroxins may be potential targets of therapeutics against liver cancer.

Acetylation of TIP60 at K104 is essential for metabolic stress-induced apoptosis in cells of hepatocellular cancer.

Tumor cells often encounter hypoglycemic microenvironment due to rapid cell expansion. It remains elusive how tumors reprogram the genome to survive the metabolic stress. The tumor suppressor TIP60 functions as the catalytic subunit of the human NuA4 histone acetyltransferase (HAT) multi-subunit complex and is involved in many different cellular processes including DNA damage response, cell growth and apoptosis. Attenuation of TIP60 expression has been detected in various tumor types. The function of TIP60 in tumor development has not been fully understood. Here we found that suppressing TIP60 inhibited p53 K120 acetylation and thus rescued apoptosis induced by glucose deprivation in hepatocellular cancer cells. Excitingly, Lys-104 (K104), a previously identified lysine acetylation site of TIP60 with unknown function, was observed to be indispensable for inducing p53-mediated apoptosis under low glucose condition. Mutation of Lys-104 to Arg (K104R) impeded the binding of TIP60 to human NuA4 complex, suppressed the acetyltransferase activity of TIP60, and inhibited the expression of pro-apoptotic genes including NOXA and PUMA upon glucose starvation. These findings demonstrate the critical regulation of TIP60/p53 pathway in apoptosis upon metabolic stress and provide a novel insight into the down-regulation of TIP60 in tumor cells.

Prioritizing Popular Proteins in Liver Cancer: Remodelling One-Carbon Metabolism.

Primary liver cancer (HCC) is recognized as the fifth most common neoplasm and the second leading cause of cancer death worldwide. Most risk factors are known, and the molecular pathogenesis has been widely studied in the past decade; however, the underlying molecular mechanisms remain to be unveiled, as they will facilitate the definition of novel biomarkers and clinical targets for more effective patient management. We utilize the B/D-HPP popular protein strategy. We report a list of popular proteins that have been highly cocited with the expression "liver cancer". Several enzymes highlight the known metabolic remodeling of liver cancer cells, four of which participate in one-carbon metabolism. This pathway is central to the maintenance of differentiated hepatocytes, as it is considered the connection between intermediate metabolism and epigenetic regulation. We designed a targeted selective reaction monitoring (SRM) method to follow up one-carbon metabolism adaptation in mouse HCC and in regenerating liver following exposure to CCl4. This method allows systematic monitoring of one-carbon metabolism and could prove useful in the follow-up of HCC and of chronically liver-diseased patients (cirrhosis) at risk of HCC. The SRM data are available via ProteomeXchange in PASSEL (PASS01060).

The anaphase promoting complex impacts repair choice by protecting ubiquitin signalling at DNA damage sites.

Double-strand breaks (DSBs) are repaired through two major pathways, homology-directed recombination (HDR) and non-homologous end joining (NHEJ). While HDR can only occur in S/G2, NHEJ can happen in all cell cycle phases (except mitosis). How then is the repair choice made in S/G2 cells? Here we provide evidence demonstrating that APCCdh1 plays a critical role in choosing the repair pathways in S/G2 cells. Our results suggest that the default for all DSBs is to recruit 53BP1 and RIF1. BRCA1 is blocked from being recruited to broken ends because its recruitment signal, K63-linked poly-ubiquitin chains on histones, is actively destroyed by the deubiquitinating enzyme USP1. We show that the removal of USP1 depends on APCCdh1 and requires Chk1 activation known to be catalysed by ssDNA-RPA-ATR signalling at the ends designated for HDR, linking the status of end processing to RIF1 or BRCA1 recruitment.

FTO Plays an Oncogenic Role in Acute Myeloid Leukemia as a N6-Methyladenosine RNA Demethylase.

N6-Methyladenosine (m6A) represents the most prevalent internal modification in mammalian mRNAs. Despite its functional importance in various fundamental bioprocesses, the studies of m6A in cancer have been limited. Here we show that FTO, as an m6A demethylase, plays a critical oncogenic role in acute myeloid leukemia (AML). FTO is highly expressed in AMLs with t(11q23)/MLL rearrangements, t(15;17)/PML-RARA, FLT3-ITD, and/or NPM1 mutations. FTO enhances leukemic oncogene-mediated cell transformation and leukemogenesis, and inhibits all-trans-retinoic acid (ATRA)-induced AML cell differentiation, through regulating expression of targets such as ASB2 and RARA by reducing m6A levels in these mRNA transcripts. Collectively, our study demonstrates the functional importance of the m6A methylation and the corresponding proteins in cancer, and provides profound insights into leukemogenesis and drug response.

Growth Arrest Specific 2 (GAS2) is a Critical Mediator of Germ Cell Cyst Breakdown and Folliculogenesis in Mice.

In the mouse ovary, the primordial follicle pool is established through a diverse array of signaling pathways and tissue remodeling events. Growth arrest specific gene two (GAS2) is a highly conserved cytoskeleton-associated protein whose in vivo function remains unclear. In Drosophila, loss of the GAS2 homolog, Pigs, results in infertility. We demonstrate herein that, in the mouse ovary, GAS2 is expressed in the stromal cells surrounding the oocyte cysts on 16.5 dpc, and in stromal cells surrounding growing follicles during juvenile and adult life. We have generated genetically engineered mice with inactivated Gas2. Gas2 homozygous mutant mice are viable but have severely impaired fertility in females, in which oocyte cyst breakdown is disrupted and follicle growth is impaired, with significantly reduced numbers of large antral follicles and corpora lutea. In these mutant mice, the organization of the basal lamina surrounding developing follicles is severely defective at multiple stages of folliculogenesis. We also found that Notch signaling activity was altered in ovaries from Gas2 null mice around the time of birth and during follicular development later in life. These results indicate that GAS2 is a critical and novel regulator of tissue remodeling in the ovary during oocyte cyst breakdown and folliculogenesis.

Hypomorphism of Fto and Rpgrip1l causes obesity in mice.

Noncoding polymorphisms in the fat mass and obesity-associated (FTO) gene represent common alleles that are strongly associated with effects on food intake and adiposity in humans. Previous studies have suggested that the obesity-risk allele rs8050136 in the first intron of FTO alters a regulatory element recognized by the transcription factor CUX1, thereby leading to decreased expression of FTO and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Here, we evaluated the effects of rs8050136 and another potential CUX1 element in rs1421085 on expression of nearby genes in human induced pluripotent stem cell-derived (iPSC-derived) neurons. There were allele-dosage effects on FTO, RPGRIP1L, and AKT-interacting protein (AKTIP) expression, but expression of other vicinal genes, including IRX3, IRX5, and RBL2, which have been implicated in mediating functional effects, was not altered. In vivo manipulation of CUX1, Fto, and/or Rpgrip1l expression in mice affected adiposity in a manner that was consistent with CUX1 influence on adiposity via remote effects on Fto and Rpgrip1l expression. In support of a mechanism, mice hypomorphic for Rpgrip1l exhibited hyperphagic obesity, as the result of diminished leptin sensitivity in Leprb-expressing neurons. Together, the results of this study indicate that the effects of FTO-associated SNPs on energy homeostasis are due in part to the effects of these genetic variations on hypothalamic FTO, RPGRIP1L, and possibly other genes.

In vitro CRISPR-Cas9-mediated efficient Ad5 vector modification.

The CRISPR-Cas9 genome editing system has been widely used in multiple cells and organisms. Here we developed a CRISPR-Cas9 based in vitro large DNA vector editing system, using the Ad5-based vector as an example. We demonstrate use of this system to generate targeted mutations, in-frame gene deletion, and gene replacement. This in vitro CRISPR editing system exhibits high efficiency and accuracy. We believe this system can be applied in a variety of experimental settings.