Abstract

Since the implementation of ATRA (all-trans retinoic acid) in therapeutic regimens for patients suffering from APL, the prognosis for this AML subtype has changed significantly in terms of both disease progression and long-term survival rates. Before the discovery of the remarkable therapeutic effects of ATRA, APL was considered the most aggressive form of leukemia, due to early death (mainly caused by fatal bleeding) and rapid progression, earning the infamous reputation of being a deadly disease. Thanks to clinical research, we now have effective targeted therapies that allow for a remarkable decrease in mortality rates and improved prognosis. These significant and encouraging results apply to the vast majority of APL patients who are sensitive to ATRA-induced differentiation therapy; however, many patients suffer from therapy resistance. While targeted therapy induces differentiation in leukemic promyelocytes and promotes favorable outcomes, it is not effective for patients who develop resistance to ATRA and do not achieve complete remission. The pathways involved in ATRA resistance in APL are not completely understood and have been the subject of numerous investigations because of their therapeutic relevance. In recent years, the roles of the pseudokinase TRIB3 and the transcription factor TWIST1 have been studied for their possible contribution to both disease progression and treatment resistance. These molecules interfere with pathways selectively exploited by targeted therapy to induce PML/RARA degradation and cell differentiation, thereby altering signaling in leukemic cells. ATRA and ATO promote leukemia regression by activating fusion protein-degradation pathways; thus, interference with this process can cause resistance to standard therapy. Early death rates also remain a major challenge to address. This mini-review aims to present the latest advances in our understanding of the molecular mechanisms—specifically focusing on TRIB3 and TWIST1—that lead to ATRA and ATO resistance, which today represent the new frontier in the fight against this disease.

Content

INTRODUCTION
Acute promyelocytic leukemia is a subset of acute myeloid leukemia, the M3 type, characterized by the translocation t(15;17). This reciprocal balanced translocation leads to the formation of the fusion protein PML::RARA by the juxtaposition of the PML (promyelocytic leukemia) gene and the RARA gene for retinoic acid receptors ¹. RARA is a transcription factor as well as a nuclear receptor for retinoic acid (retinoids have been shown to play a role in hematopoietic differentiation), while PML is a tumor suppressor involved in the regulation of cell proliferation. The fusion protein resulting from these two genes, PML::RARA, plays a pivotal role in acute promyelocytic leukemia APL pathogenesis by inhibiting myeloid differentiation at the promyelocyte stage, thereby leading to leukemic cell accumulation in the bone marrow. PML::RARA forms a homodimer that disrupts wild-type RARA physiological pathways by negatively regulating the transcription of genes involved in hematopoiesis and cell differentiation. PML normally acts as a tumor suppressor and regulates apoptosis via caspase signaling activation and senescence via the p53 pathway. These regulatory mechanisms are compromised by the fusion protein PML::RARA, which disrupts PML nuclear bodies ². Because of its driver role in almost all cases of APL, PML::RARA is considered a diagnostic marker. However, some variants of AML have been reported to present different translocations involving RARA and other genes, combined with additional mutations. In the 1990s, the implementation of ATRA in first-line therapeutic regimens for APL revolutionized patients outcomes, turning this once-fatal disease into one with a favorable prognosis in most cases. ATRA for APL has since become the paradigm of targeted therapies and paved the way for many treatments selectively designed to regulate specific molecular mechanisms underlying tumorigenesis. This pharmacological therapy, often combined with chemotherapy and ATO (arsenic trioxide), induces differentiation in myeloid cells as well as disease regression by directly targeting PML::RARA and promoting its degradation ³. ATRA and ATO promote APL regression by up-regulating degradation of PML::RARA through autophagy, UPS, caspase-like signaling, and sumoylation. Their efficacy has significantly improved survival rates, but some patients remain resistant, showing no differentiation and persistent PML::RARA expression because the pathways normally activated by ATRA are compromised. PML::RARA degradation, achieved via UPS, autophagy, sumoylation, and caspase-like pathways ⁴, is a key therapeutic mechanism ^3,5-7, and its inhibition is closely associated with APL progression and therapy resistance. Importantly, PML::RARA degradation via ATRA treatment is not only associated with blast differentiation but also with depletion of leukemia-initiating cells (LICs). LIC loss is essential for leukemia eradication ⁸. Mutations in the RA-binding domain of the fusion protein and altered ATRA metabolism, which prevent intracellular concentrations of ATRA from reaching therapeutic levels, have also been described as possible causes of acquired therapy resistance ⁹. Recent studies have focused on the pseudokinase TRIB3 and transcription factor TWIST1, both found at higher concentrations in APL cells compared to other AML subtypes, and both implicated in APL pathogenesis and resistance. TRIB3 promotes disease progression and therapy resistance by stabilizing PML::RARA and inhibiting its degradation. It interacts with p62 (an autophagy substrate), while other TRIBBLES family members such as TRIB1 and TRIB2 disrupt expression of transcription factor CEBPalpha ^10-12. TWIST1, an EMT-related transcription factor, promotes extra-medullary infiltration in AML ¹³ and tumor metastasis ¹⁴. Its high concentration is maintained by TRIB3, which prevents TWIST1 degradation by altering ubiquitination signaling. Knockdown of TRIB3 decreases TWIST1, enabling PML::RARA degradation and disease regression. TWIST1 expression supports survival ¹³, proliferation ¹⁵, and therapy resistance in leukemic promyelocytes, specifically by interfering with PML::RARA degradation ¹³. Significant evidence points to an interplay between TRIB3 and TWIST1 in promoting pathogenesis and ATRA resistance, through both direct and indirect mechanisms ². Their combined actions stabilize PML::RARA, preventing its degradation and cell differentiation. We will present the molecular pathways by which their interaction mediates therapeutic resistance and explore how these adverse effects may be reversed to enable new strategies.
II. THE ROLE OF TRIB3 IN APL PATHOGENESIS AND ATRA RESISTANCE
II.1 MOLECULAR PATHWAYS CONTROLLED BY TRIB3 IN APL CELLS
TRIB3, a member of the Tribbles family, is a pseudokinase involved in a plethora of molecular pathways in the cell. These pseudokinases act as intracellular stress sensors and play a role in inflammation signaling as well as in cancer (not necessarily just leukemia ¹⁶). TRIB1 and TRIB2 are shown to promote leukemic progression by interfering with CEBPa expression, a myeloid differentiation transcription factor; however TRIB3 is not involved in this specific pathway. The precise role of TRIB3 in APL pathogenesis has only recently been discovered, and while the other proteins of this family inhibit myeloid differentiation, this pseudokinase interacts directly with fusion protein PML::RARA, regulating its expression by affecting the pathways related to its degradation in APL cells. PML::RARA is a key genetic driver of this type of leukemia, directly dysregulating cell differentiation, and alterations of its clearance could lead to treatment resistance. High levels of TRIB3 have been found in APL-affected cells, demonstrating its role in both pathogenesis and treatment resistance ². TRIB3 stabilizes fusion protein by inhibiting its sumoylation, ubiquitination, and autophagy-related degradation pathways. PML::RARA disrupts PML-NB formation, compromising p53 signaling. TRIB3 affects senescence, differentiation, and self-renewal programs and interacts with the autophagy substrate p62, altering ubiquitin-related autophagy of PML::RARA. Alteration of UPS and autophagy by this pseudokinase has oncogenic effects in various cancers. TRIB3 regulates these pathways, promoting pathogenesis and inhibiting PML::RARA degradation ^13,17,18.
II.2 TRIB3 INHIBITS PML/RARA DEGRADATION
TRIB3 promotes APL progression by stabilizing the fusion protein PML::RARA ^2,12,13. High levels of this pseudokinase physically bind to the chimeric protein, inhibiting its degradation. PML::RARA degradation is a fundamental therapeutic strategy achieved by ATRA and ATO treatment, inducing cell differentiation and disease regression in sensitive patients. Inhibition of PML::RARA degradation leads to APL progression and inhibits differentiation. High TRIB3 levels inhibit ubiquitination and sumoylation of PML::RARA as well as PML-NB assembly, promoting disease progression. TRIB3 binding also disrupts UPS signaling and autophagy flux by interacting with p62. TRIB3 depletion restores PML::RARA degradation and ATRA-induced therapeutic effects.
II.3 TRIB3 BINDS TO PML DOMAINS OF PML/RARA TO INHIBIT ITS SUMOYLATION
A study ² showed that TRIB3 mainly binds to B-Box1 and Ring-finger motifs of the PML-domain in PML::RARA but can also bind the full protein or NLS-domains. These domains contain lysine residues necessary for sumoylation (B-Box1: K160; RING-finger: K65; NLS: K490/497). This binding inhibits sumoylation, disrupts PML-NB assembly, and impairs p53 signaling, promoting pathogenesis. TRIB3 also prevents PML::RARA binding with E2 and E3 enzymes. Specific peptides, such as Pep2-S160, that target the TRIB3-PML interaction increase PML::RARA degradation, restore PML-NB assembly, and enhance ATRA and ATO effects. Notably, ATRA and ATO increase TRIB3 levels in APL, providing a potential mechanism for resistance.

Figure 1. TRIB3 interacts with PML::RARA to inhibit its sumoylation.
(1)TRIB3 physically binds with PML-domain of fusion protein PML::RARA at its sumoylation sites. (2) TRIB3 binding with PML::RARA inhibits PML::RARA sumoylation and degradation, which is a specific ATRA-induced therapeuitic strategy.

II.4 TRIB3 INHIBITS PML/RARA CLEARANCE BY INTERACTING WITH p62 AND INHIBITING AUTOPHAGY
Autophagy and UPS are primary pathways activated by ATRA and ATO to clear PML::RARA ¹⁹. ATRA induces autophagy-mediated degradation by silencing mTOR signaling; rapamycin enhances PML::RARA degradation and therapeutic effects. Autophagy inhibitors reduce clearance, and ATRA fails in resistant cells ²⁰. The autophagy substrate p62 binds ubiquitinated proteins such as PML::RARA and directs them to autophagy ^19,21. Matrine (MAT) reverses ATRA resistance by regulating ubiquitination and autophagy, especially when combined with rapamycin ^20,22. Knockdown of p62 disrupts PML::RARA degradation ²². TRIB3 interacts with p62, leading to its accumulation and functional loss, preventing binding to ubiquitinated PML::RARA, thereby inhibiting autophagy and contributing to resistance ²³.
III. TWIST1 PROMOTES PRO-ONCOGENIC MOLECULAR PATHWAYS IN APL AFFECTED CELLS
III.1 TWIST1 IS HIGHLY EXPRESSED IN APL
APL cells exhibit high TWIST1, a bHLH transcription factor promoting epithelial-mesenchymal transition. EMT-TFs are detectable in AML subtypes but overexpressed in APL. TWIST1 is involved in embryogenesis but promotes metastasis and extra-medullary infiltration in AML, contributing to aggressive disease and high early death rates due to hemorrhage. High TWIST1 inhibits PML::RARA degradation, supporting proliferation, stem-like behavior, chemoresistance, and adverse prognosis ^15,24.
III.2 TWIST1 PROMOTES APL PROGRESSION BY INHIBITING PML/RARA DEGRADATION
EMT-TFs promote oncogenesis by inhibiting p53 signaling, supporting proliferation, and invasion ^25,26. TWIST1 colocalizes partially with PML::RARA in APL cell lines, indirectly stabilizing it. PML::RARA knockdown reduces TWIST1, and TWIST1 knockdown decreases PML::RARA, promoting apoptosis and cell differentiation ¹³. TWIST1 prevents PML::RARA degradation, supporting leukemic progression.
III.3 TRIB3 INHIBITS TWIST1 DEGRADATION BY DISRUPTING ITS UBIQUITINATION
TWIST1 is ubiquitinated and degraded because its transcription is not directly controlled by PML::RARA. TRIB3 colocalizes with TWIST1 and prevents its ubiquitination and degradation, maintaining its pro-oncogenic activity. TRIB3 knockdown increases TWIST1 ubiquitination and reduces PML::RARA levels ^13,27.

Figure2. TRIB3 binds with TWIST1 to inhibit its degradation, preventing PML/RARA degradation. (1) C-terminal domain of TRIB3 binds with WR domain of TWIST1. (2) TRIB3-TWIST1 binding inhibits TWIST1 ubiquitination. (3) TRIB3-TWIST1 promotes high TWIST1 levels. (4) High TWIST1 levels allow for PML::RARA overexpression by inhibiting its degradation.


IV. TRIB3 AND TWIST1 BINDING PROMOTES ATRA RESISTANCE IN APL AFFECTED CELLS
TRIB3 stabilizes TWIST1 via C-terminal – WR domain binding, preventing ubiquitination ^13,28. This interaction inhibits UPS- and p62-mediated degradation pathways, maintaining pro-oncogenic activity. TRIB3 and TWIST1 levels decrease in ATRA-sensitive cells but not in resistant cells. Knockdown of TRIB3 restores TWIST1 degradation, PML::RARA clearance, and cell differentiation, while peptide-mediated disruption of TRIB3-TWIST1 binding enhances apoptosis ¹³.
V. TARGETING TRIB3 AND INTERACTION BETWEEN TRIB3 AND TWIST1 AS A NEW THERAPEUTIC STRATEGY FOR HIGH-RISK PATIENTS AND ATRA RESISTANCE
Inhibition of TRIB3 promotes apoptosis and differentiation, reversing ATRA resistance by restoring autophagic pathways. Targeting TRIB3-PML/RARA binding with peptides increases PML::RARA degradation and ATRA sensitivity. Targeting TRIB3-TWIST1 interaction also promotes TWIST1 degradation, reduces extra-medullary infiltration, leukemic stem cell formation, and early death rates. These strategies could extend favorable outcomes to patients that display therapy-resistance ^2,13,27.
CONCLUSIONS
This mini-review summarizes the roles of TRIB3 and TWIST1 in ATRA resistance in APL. Both individually and via their interaction, they stabilize PML::RARA and inhibit its degradation, interfering with ATRA and ATO therapeutic mechanisms. Understanding these molecular pathways allows for the design of new strategies to restore ATRA sensitivity and improve patient outcomes. Targeting TRIB3, TWIST1, and their interactions may overcome resistance, reduce early deaths, and offer new therapeutic avenues to help all patients who courageously fight against APL.