Structural Basis of PML-RARA Oncoprotein Targeting by Arsenic Unravels a Cysteine Rheostat Controlling PML Body Assembly and Function.

PML nuclear bodies (NB) are disrupted in PML-RARA-driven acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) cures 70% of patients with APL, driving PML-RARA degradation and NB reformation. In non-APL cells, arsenic binding onto PML also amplifies NB formation. Yet, the actual molecular mechanism(s) involved remain(s) elusive. Here, we establish that PML NBs display some features of liquid-liquid phase separation and that ATO induces a gel-like transition. PML B-box-2 structure reveals an alpha helix driving B2 trimerization and positioning a cysteine trio to form an ideal arsenic-binding pocket. Altering either of the latter impedes ATO-driven NB assembly, PML sumoylation, and PML-RARA degradation, mechanistically explaining clinical ATO resistance. This B2 trimer and the C213 trio create an oxidation-sensitive rheostat that controls PML NB assembly dynamics and downstream signaling in both basal state and during stress response. These findings identify the structural basis for arsenic targeting of PML that could pave the way to novel cancer drugs. SIGNIFICANCE: Arsenic curative effects in APL rely on PML targeting. We report a PML B-box-2 structure that drives trimer assembly, positioning a cysteine trio to form an arsenic-binding pocket, which is disrupted in resistant patients. Identification of this ROS-sensitive triad controlling PML dynamics and functions could yield novel drugs. See related commentary by Salomoni, p. 2505. This article is featured in Selected Articles from This Issue, p. 2489.

The landscape of novel strategies for acute myeloid leukemia treatment: Therapeutic trends, challenges, and future directions.

Acute myeloid leukemia (AML) is a highly heterogeneous subtype of hematological malignancies with a wide spectrum of cytogenetic and molecular abnormalities, which makes it difficult to manage and cure. Along with the deeper understanding of the molecular mechanisms underlying AML pathogenesis, a large cohort of novel targeted therapeutic approaches has emerged, which considerably expands the medical options and changes the therapeutic landscape of AML. Despite that, resistant and refractory cases caused by genomic mutations or bypass signalling activation remain a great challenge. Therefore, discovery of novel treatment targets, optimization of combination strategies, and development of efficient therapeutics are urgently required. This review provides a detailed and comprehensive discussion on the advantages and limitations of targeted therapies as a single agent or in combination with others. Furthermore, the innovative therapeutic approaches including hyperthermia, monoclonal antibody-based therapy, and CAR-T cell therapy are also introduced, which may provide safe and viable options for the treatment of patients with AML.

Hyperthermia promotes degradation of the acute promyelocytic leukemia driver oncoprotein ZBTB16/RARα.

The acute promyelocytic leukemia (APL) driver ZBTB16/RARα is generated by the t(11;17) (q23;q21) chromosomal translocation, which is resistant to combined treatment of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) or conventional chemotherapy, resulting in extremely low survival rates. In the current study, we investigated the effects of hyperthermia on the oncogenic fusion ZBTB16/RARα protein to explore a potential therapeutic approach for this variant APL. We showed that Z/R fusion protein expressed in HeLa cells was resistant to ATO, ATRA, and conventional chemotherapeutic agents. However, mild hyperthermia (42 °C) rapidly destabilized the ZBTB16/RARα fusion protein expressed in HeLa, 293T, and OCI-AML3 cells, followed by robust ubiquitination and proteasomal degradation. In contrast, hyperthermia did not affect the normal (i.e., unfused) ZBTB16 and RARα proteins, suggesting a specific thermal sensitivity of the ZBTB16/RARα fusion protein. Importantly, we found that the destabilization of ZBTB16/RARα was the initial step for oncogenic fusion protein degradation by hyperthermia, which could be blocked by deletion of nuclear receptor corepressor (NCoR) binding sites or knockdown of NCoRs. Furthermore, SIAH2 was identified as the E3 ligase participating in hyperthermia-induced ubiquitination of ZBTB16/RARα. In short, these results demonstrate that hyperthermia could effectively destabilize and subsequently degrade the ZBTB16/RARα fusion protein in an NCoR-dependent manner, suggesting a thermal-based therapeutic strategy that may improve the outcome in refractory ZBTB16/RARα-driven APL patients in the clinic.

The Landscape of Nucleic-Acid-Based Aptamers for Treatment of Hematologic Malignancies: Challenges and Future Directions.

Hematologic malignancies, including leukemia, lymphoma, myeloproliferative disorder and plasma cell neoplasia, are genetically heterogeneous and characterized by an uncontrolled proliferation of their corresponding cell lineages in the bone marrow, peripheral blood, tissues or plasma. Although there are many types of therapeutic drugs (e.g., TKIs, chemotherapy drugs) available for treatment of different malignancies, the relapse, drug resistance and severe side effects due to the lack of selectivity seriously limit their clinical application. Currently, although antibody-drug conjugates have been well established as able to target and deliver highly potent chemotherapy agents into cancer cells for the reduction of damage to healthy cells and have achieved success in leukemia treatment, they still also have shortcomings such as high cost, high immunogenicity and low stability. Aptamers are ssDNA or RNA oligonucleotides that can also precisely deliver therapeutic agents into cancer cells through specifically recognizing the membrane protein on cancer cells, which is similar to the capabilities of monoclonal antibodies. Aptamers exhibit higher binding affinity, lower immunogenicity and higher thermal stability than antibodies. Therefore, in this review we comprehensively describe recent advances in the development of aptamer-drug conjugates (ApDCs) with cytotoxic payload through chemical linkers or direct incorporation, as well as further introduce the latest promising aptamers-based therapeutic strategies such as aptamer-T cell therapy and aptamer-PROTAC, clarifying their bright application, development direction and challenges in the treatment of hematologic malignancies.

m6A modification confers thermal vulnerability to HPV E7 oncotranscripts via reverse regulation of its reader protein IGF2BP1 upon heat stress.

Human papillomavirus (HPV)-induced carcinogenesis critically depends on the viral early protein 7 (E7), making E7 an attractive therapeutic target. Here, we report that the E7 messenger RNA (mRNA)-containing oncotranscript complex can be selectively targeted by heat treatment. In HPV-infected cells, viral E7 mRNA is modified by N6-methyladenosine (m6A) and stabilized by IGF2BP1, a cellular m6A reader. Heat treatment downregulates E7 mRNA and protein by destabilizing IGF2BP1 without the involvement of canonical heat-shock proteins and reverses HPV-associated carcinogenesis in vitro and in vivo. Mechanistically, heat treatment promotes IGF2BP1 aggregation only in the presence of m6A-modified E7 mRNA to form distinct heat-induced m6A E7 mRNA-IGF2BP1 granules, which are resolved by the ubiquitin-proteasome system. Collectively, our results not only show a mutual regulation between m6A RNA and its reader but also provide a heat-treatment-based therapeutic strategy for HPV-associated malignancies by specifically downregulating E7 mRNA-IGF2BP1 oncogenic complex.

Linear and Circular Long Non-Coding RNAs in Acute Lymphoblastic Leukemia: From Pathogenesis to Classification and Treatment.

The coding regions account for only a small part of the human genome, and the remaining vast majority of the regions generate large amounts of non-coding RNAs. Although non-coding RNAs do not code for any protein, they are suggested to work as either tumor suppressers or oncogenes through modulating the expression of genes and functions of proteins at transcriptional, posttranscriptional and post-translational levels. Acute Lymphoblastic Leukemia (ALL) originates from malignant transformed B/T-precursor-stage lymphoid progenitors in the bone marrow (BM). The pathogenesis of ALL is closely associated with aberrant genetic alterations that block lymphoid differentiation and drive abnormal cell proliferation as well as survival. While treatment of pediatric ALL represents a major success story in chemotherapy-based elimination of a malignancy, adult ALL remains a devastating disease with relatively poor prognosis. Thus, novel aspects in the pathogenesis and progression of ALL, especially in the adult population, need to be further explored. Accumulating evidence indicated that genetic changes alone are rarely sufficient for development of ALL. Recent advances in cytogenic and sequencing technologies revealed epigenetic alterations including that of non-coding RNAs as cooperating events in ALL etiology and progression. While the role of micro RNAs in ALL has been extensively reviewed, less attention, relatively, has been paid to other non-coding RNAs. Herein, we review the involvement of linear and circular long non-coding RNAs in the etiology, maintenance, and progression of ALL, highlighting the contribution of these non-coding RNAs in ALL classification and diagnosis, risk stratification as well as treatment.

Aptamers: an emerging navigation tool of therapeutic agents for targeted cancer therapy.

Chemotherapeutic agents have been used for the treatment of numerous cancers, but due to poor selectivity and severe systemic side effects, their clinical application is limited. Single-stranded DNA (ssDNA) or RNA aptamers could conjugate with highly toxic chemotherapy drugs, toxins, therapeutic RNAs or other molecules as novel aptamer-drug conjugates (ApDCs), which are capable of significantly improving the therapeutic efficacy and reducing the systemic toxicity of drugs and have great potential in clinics for targeted cancer therapy. In this review, we have comprehensively discussed and summarized the current advances in the screening approaches of aptamers for specific cancer biomarker targeting and development of the aptamer-drug conjugate strategy for targeted drug delivery. Moreover, considering the huge progress in artificial intelligence (AI) for protein and RNA structure predictions, automatic design of aptamers using deep/machine learning techniques could be a powerful approach for rapid and precise construction of biopharmaceutics (i.e., ApDCs) for application in cancer targeted therapy.

Hyperthermia Selectively Destabilizes Oncogenic Fusion Proteins.

The PML/RARα fusion protein is the oncogenic driver in acute promyelocytic leukemia (APL). Although most APL cases are cured by PML/RARα-targeting therapy, relapse and resistance can occur due to drug-resistant mutations. Here we report that thermal stress destabilizes the PML/RARα protein, including clinically identified drug-resistant mutants. AML1/ETO and TEL/AML1 oncofusions show similar heat shock susceptibility. Mechanistically, mild hyperthermia stimulates aggregation of PML/RARα in complex with nuclear receptor corepressors leading to ubiquitin-mediated degradation via the SIAH2 E3 ligase. Hyperthermia and arsenic therapy destabilize PML/RARα via distinct mechanisms and are synergistic in primary patient samples and in vivo, including three refractory APL cases. Collectively, our results suggest that by taking advantage of a biophysical vulnerability of PML/RARα, thermal therapy may improve prognosis in drug-resistant or otherwise refractory APL. These findings serve as a paradigm for therapeutic targeting of fusion oncoprotein-associated cancers by hyperthermia. SIGNIFICANCE: Hyperthermia destabilizes oncofusion proteins including PML/RARα and acts synergistically with standard arsenic therapy in relapsed and refractory APL. The results open up the possibility that heat shock sensitivity may be an easily targetable vulnerability of oncofusion-driven cancers.See related commentary by Wu et al., p. 300.

Arsenic trioxide targets Hsp60, triggering degradation of p53 and survivin.

The mechanisms of action of arsenic trioxide (ATO), a clinically used drug for the treatment of acute promyelocytic leukemia (APL), have been actively studied mainly through characterization of individual putative protein targets. There appear to be no studies at a system level. Herein, we integrate metalloproteomics through a newly developed organoarsenic probe, As-AC (C20H17AsN4O3S2) with quantitative proteomics, allowing 37 arsenic binding and 250 arsenic regulated proteins to be identified in NB4, a human APL cell line. Bioinformatics analysis reveals that ATO disrupts multiple physiological processes, in particular, chaperone-related protein folding and cellular response to stress. Furthermore, we discover heat shock protein 60 (Hsp60) as a vital target of ATO. Through biophysical and cell-based assays, we demonstrate that ATO binds to Hsp60, leading to abolishment of Hsp60 refolding capability. Significantly, the binding of ATO to Hsp60 disrupts the formation of Hsp60-p53 and Hsp60-survivin complexes, resulting in degradation of p53 and survivin. This study provides significant insights into the mechanism of action of ATO at a systemic perspective, and serves as guidance for the rational design of metal-based anticancer drugs.

Advances on molecular mechanism of hepatitis B virus-induced hepatocellular carcinoma.

The pathogenesis of hepatitis B virus （HBV）-associated hepatocellular carcinoma （HCC） is complicated with the crosstalk of multiple factors and the multi-step processes. The main mechanisms underlying the HBV-induced HCC include:①integration of HBV DNA into the host hepatocyte genome to alter gene function at the insertion site，resulting in host genome instability and expression of carcinogenic truncated proteins;②HBV gene mutations at S，C，and X coding regions in the genome;③HBV X gene-encoded HBx protein activates proto-oncogenes and inhibits tumor suppressor genes，leading to the HCC occurrence. In this article，the recent research progress on the molecular mechanism of HBV-induced HCC is comprehensively reviewed，so as to provide insights into the prevention，early prediction and postoperative adjuvant therapy of HCC.

Synthesis and characterization of CD133 targeted aptamer-drug conjugates for precision therapy of anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is an undifferentiated and highly aggressive type of thyroid cancer and is extremely resistant to standard therapies such as surgical resection and radioactive iodine therapy. Although targeted therapeutic agents including small molecule drugs and monoclonal antibodies are rapidly developed in recent years, no ATC targeted drugs are available to date; thereby, novel targeted therapies are needed to improve the outcomes of ATC patients. Aptamers are single-stranded DNA (or RNA) molecules that can selectively bind to cancer specific antigens, and aptamer-based targeted therapy has certain advantages over that based on antibodies due to its high binding affinity and low immunogenicity. Here, we identified that CD133, a cancer stem cell marker, was specifically expressed in ATC tumor tissues and cells, implying that CD133 is a potential drug target for ATC therapy. Additionally, we successfully obtained a CD133 targeted aptamer AP-1 by paired cell-based SELEX, which can precisely recognize CD133 antigen in vitro. Furthermore, the truncated AP-1-M aptamer from its precursor AP-1 has shown higher binding affinity for CD133, and specifically accumulated in anaplastic thyroid cancer FRO cell derived tumor in vivo. Conjugation of truncated AP-1-M with doxorubicin could dramatically inhibit CD133 positive FRO cell proliferation, induce cell apoptosis in vitro, and also suppress tumor growth in FRO cell xenograft mice in vivo. Our results clearly demonstrated that the CD133 targeted aptamer AP-1-M conjugated with anticancer drugs has potential to become a promising therapeutic approach against ATC in the near future.

Balance between the toxicity and anticancer activity of arsenic trioxide in treatment of acute promyelocytic leukemia.

Arsenic trioxide (ATO) has a long history and it is recognized as both poison and drug for more than two thousand years. Since the establishment of ATO as a frontline therapeutic agent for acute promyelocytic leukemia (APL), the survival of APL patients have been greatly improved and APL is turned from highly fatal to highly curable disease. Mechanistically, ATO can induce PML/RARα fusion protein degradation, causing APL cell differentiation and apoptosis. On the other hand, the side effects such as differentiation syndrome, cardiac conduction abnormalities and liver toxicity are often observed during the ATO treatment of APL in clinic. It is likely that the therapeutic and adverse effects of ATO is probably associated with its distinct pattern of metabolism and direct or indirect effects on different organs. In this review, we provided a comprehensive and in-depth elaboration of the cytotoxic mechanisms of ATO and its methylated metabolites based on in vivo or in vitro studies, trying to clarify the importance of achieving balance between the toxicity and anti-leukemic activity of ATO in APL treatment.

Arsenic induced epigenetic changes and relevance to treatment of acute promyelocytic leukemia and beyond.

Epigenetic alterations regulate gene expression without changes in the DNA sequence. It is well-demonstrated that aberrant epigenetic changes contribute to the leukemogenesis of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) is one of the most common drugs used in the frontline treatment of APL that act through targeting and destabilizing the PML/RARα oncofusion protein. ATO together with all-trans retinoic acid (ATRA) lead to durable remission of more than 90% non-high-risk APL patients, turning APL treatment into a paradigm of oncoprotein targeted cure. Although relapse and drug resistance in APL are yet to be resolved in the clinic, epigenetic machineries might hold the key to address this issue. Further, ATO also showed promising anticancer activities against a variety of malignancies, but its application is particularly restricted due to limited understanding of the mechanism. Thus, a thorough understanding of epigenetic mechanism behind anti-leukemic effects of ATO would benefit the development of ATO-based anticancer strategy. Role of ATRA on APL associated epigenetic alterations has been extensively studied and reviewed. Recently, accumulating evidence suggest that ATO also induces some epigenetic changes that might favor APL eradication. In this article, we comprehensively discuss arsenic induced epigenetic changes and its relevance in APL treatment and beyond, so as to provide novel insights into overcoming arsenic resistance in APL and promote application of this drug to other malignancies.

Biotransformation of arsenic trioxide by AS3MT favors eradication of acute promyelocytic leukemia: revealing the hidden facts.

Arsenic trioxide (ATO) is one of the most effective drugs for treatment of acute promyelocytic leukemia (APL). It could specifically target the PML/RARα fusion oncoprotein stability and induces APL cell differentiation as well as apoptosis. Although many studies have been conducted to document the anticancer effects and mechanism of ATO, there is little information about the association between biotransformation of ATO to active arsenic metabolites and APL therapy. Generally, ATO can be rapidly converted into trivalent methylated metabolites by arsenic (+3 oxidation state) methyltransferase (AS3MT) mostly in liver and redistributed to bloodstream of APL patients who receiving ATO treatment, thereby leading to a balance between cytotoxicity and differentiation, which is proposed to be the key event in successful treatment of APL. In this review, we comprehensively discussed possible roles of AS3MT and methylated arsenic metabolites in APL therapy, so as to reveal the association between individual differences of AS3MT expression and activity with the therapeutic efficacy of ATO in APL patients.

Therapeutic strategy of arsenic trioxide in the fight against cancers and other diseases.

Arsenic trioxide (ATO) has been recognized as a drug for the treatment of various diseases in traditional medicine for more than two thousand years. Although ATO has recently shown excellent efficacy for the treatment of acute promyelocytic leukemia (APL), it could not provide satisfactory outcomes as a single-agent for the management of non-APL leukemia or different solid tumors. Nevertheless, combination treatment strategies, e.g., ATO with other agents, have shown promising results against different diseases. Here, we introduce in depth the latest evidence and detailed insights into ATO-mediated cures for APL by targeting PML/RARα chimeric protein, followed by the preclinical and clinical efficacy of ATO on various non-APL malignancies and solid tumors. Likewise, the antiviral activity of ATO against human immunodeficiency virus (HIV) and hepatitis C virus (HCV) was also discussed briefly. Our review would provide a clear prospect for the combination of ATO with other agents for treatment of numerous neoplastic diseases, and open a new era in the clinically applicable range of arsenicals.

Involvement of PML-I in reformation of PML nuclear bodies in acute promyelocytic leukemia cells by leptomycin B.

Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation between chromosomes 15 and 17, t(15;17), resulting in the expression of PML-RARα fusion protein, which disrupts the normal PML nuclear bodies (PML-NBs) to micro-speckled pattern, leading to loss of their original functions. Moreover, reformation of PML-NBs in APL by arsenic is considered as one of the important step for APL treatment. Leptomycin B (LMB), a nuclear export inhibitor, is commonly used to inhibit the proteins exporting from the nucleus to the cytoplasm. In the present study, we found that LMB could induce the reformation of PML-NBs in leukemia NB4 cells as well as in APL blast cells from the patients, implying that nuclear shuttle proteins might be involved in the reformation of PML-NBs. Herein, we further found that LMB totally lost the ability to induce PML-NBs reformation when the endogenous PML gene was knocked out, indicating that endogenous PML protein is probably involved in the reformation of PML-NBs. More interestingly, among all PML isoforms (i.e., seven isoforms), reformation of PML-NBs was only observed when co-transfection of PML-RARα with PML-I after LMB treatment. Similarly, deletion of nuclear export signal (NES) of PML-I could also reform PML-NBs, suggesting that the protein level of endogenous PML-I in nucleus is important for the reformation of PML-NBs that interfered by PML-RARα fusion protein. Additionally, LMB has synergistic effect with iAsIII on enhancing PML-RARα fusion protein degradation, and it might provide new insight into APL treatment at clinical level in the near future.

Irreversibility of arsenic trioxide induced PML/RARα fusion protein solubility changes.

Arsenic trioxide (As2O3) is one of the most effective drugs for the treatment of acute promyelocytic leukemia (APL), and induces the degradation of chimeric oncoprotein PML/RARα (P/R) and APL cell differentiation. Recent evidence has suggested that P/R fusion protein degradation by arsenic occurs through two steps, namely, rapid solubility change/shift of the P/R fusion protein following arsenic treatment (i.e., transfer of P/R protein from the soluble fraction to the insoluble pellet fraction), and subsequent degradation of these insoluble proteins. However, there is little information regarding the reversibility of arsenic induced P/R fusion protein solubility change as well as protein degradation in the insoluble fraction after removing arsenic. In this study, we used APL cell line NB4 or P/R and PML over-expressed 293T cells as well as HeLa cells to reveal the solubility change of P/R and PML by arsenic exposure, and further determined the fate of these insoluble proteins after the removal of arsenic. Here, for the first time, we found that arsenic induced P/R or PML protein solubility change is an irreversible process. Once arsenic induces a P/R or PML protein solubility change, these insoluble proteins could be degraded by the proteasomal pathway even without continuous arsenic treatment. However, PML and P/R proteins can be newly synthesized after the removal of arsenic, suggesting that great caution should be taken in the clinical therapy of APL patients before ending arsenic treatment.