Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.     

NLS-RARα contributes to differentiation block and increased leukemogenic potential in vivo

The fusion oncogene, promyelocytic leukemia (PML)-retinoic acid receptor-α (RARα), is crucial for acute promyelocytic leukemia (APL) pathogenesis. Previous studies have reported that PML-RARα is cleaved by neutrophil elastase (NE), an early myeloid-specific serine protease, leading to translocation of the nuclear localization signal (NLS) of the PML protein to the N-terminal of RARα. This study was designed to evaluate the value of NLS-RARα in the early diagnosis of APL. To investigate the potential functional role of NLS-RARα in leukemogenesis, HL-60 and U937 cell lines were transfected with NLS-RARα lentivirus and negative control (LVNC). The results showed that the induced expression of NLS-RARα down-regulated expressions of CD11b, CD11c, and CD14 compared to the LVNC group induced by 1α, 25-dihydroxyvitamin D₃(1,25(OH)₂D₃). This suggested that NLS-RARα overexpression inhibited granulocytic and monocytic differentiation of myeloid leukemia cells. In addition, Wright-Giemsa staining, flow cytometry, respiratory burst assay, and NBT reduction assay all confirmed the importance of NLS-RARα in differentiation. The mechanistic investigations revealed that induced NLS-RARα expression inhibited 1,25(OH)₂D₃-induced granulocytic differentiation by regulating the cell cycle regulators p19^INK4D, p21^WAF1/CIP1, cyclinD1, cyclin E1, and pRB. Furthermore, the cleaved protein NLS-RARα enhanced the oncogenicity of U937 cells in NOD/SCID mice. These findings collectively demonstrated that NLS-RARα blocked granulocytic and monocytic differentiation of myeloid leukemia cells by inhibiting the downstream targets of the RARα signal pathway and the cell cycle. This may provide a promising new target and method for diagnosing and treating APL.     

Cloning of the murine homolog of the leukemia-associated PML gene

PML, a Ring-finger protein, participates in the disruption of normal myeloid differentiation when fused to the retinoic acid receptor alpha (RAR) by the translocation between Chromosomes (Chrs) 15 and 17 in acute promyelocytic leukemia (APL). As an initial step in the characterization of PML in species other than human, a murine cDNA clone of the PML gene was isolated and sequenced, and the intron/exon organization of the murine locus determined. The predicted amino acid sequence of the mouse PML protein shows 80% similarity to that of its human homolog. However, the mouse and human proteins show greater than 90% similarity in the proposed functional domains of the proteins. Despite its role in the etiology of APL, PML expression is not detectably altered during granulocytic differentiation in a murine in vitro system. Chromosomal localization of the Pml locus by somatic cell hybrids and by linkage analysis indicates that the gene maps to a region of mouse Chr 9 with known linkage homology to the region on human Chr 15q to which PML has been localized.     

PRAM-1 is required for optimal integrin-dependent neutrophil function

PML-retinoic acid receptor alpha (RARalpha) regulated adaptor molecule 1 (PRAM-1) is an intracellular adaptor molecule that is upregulated during the induced granulocytic differentiation of promyelocytic leukemic cells and during normal human myelopoiesis. This report describes the generation of PRAM-1-deficient mice and an analysis of the function of this adaptor in neutrophil differentiation and mature neutrophil function. We demonstrate here that neutrophil differentiation is not impaired in PRAM-1-deficient mice and that PRAM-1-deficient neutrophils function normally following engagement of Fcgamma receptors. In contrast, mature PRAM-1-null neutrophils exhibit significant defects in adhesion-dependent reactive oxygen intermediate production and degranulation. Surprisingly, other integrin-dependent responses, such as cell spreading and activation of several signaling pathways, are normal. Together, these findings demonstrate the uncoupling of key integrin-dependent responses in the absence of PRAM-1 and show this adaptor to be critical for select integrin functions in neutrophils.     

Leukemia: beneficial actions of retinoids and rexinoids

Acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia, is the prototype of a cancer that can be cured by differentiation therapy using combined retinoic acid (RA) and chemotherapy. Acute promyelocytic leukemia is caused by chromosomal translocations, which in the large majority of cases generate the prototypic promyelocytic leukemia-retinoic-acid receptor alpha (PML-RARalpha) an oncogenic fusion protein formed from the retinoic-acid receptor alpha and the so-called PML protein. The fusion protein leads to the deregulation of wild type PML and RARalpha function, thus inducing the differentiation block and an altered survival capacity of promyelocytes of affected patients. A plethora of studies have revealed molecular details that account for the oncogenic properties of acute promyelocytic leukemia fusion proteins and the events that contribute to the therapy-induced differentiation and apoptosis of patients' blasts. Illustrating the beneficial mechanisms of action of retinoids for acute promyelocytic leukemia patients this review goes on to discuss a plethora of recently recognized molecular paradigms by which retinoids and rexinoids, alone or in combination with other compounds, regulate growth, differentiation and apoptosis also in non-acute promyelocytic leukemia cells, highlighting their potential as drugs for cancer therapy and prevention.