Association of leukemic molecular profile with efficacy of inotuzumab ozogamicin in adults with relapsed/refractory ALL.

The phase 3 INO-VATE trial demonstrated higher rates of remission, measurable residual disease negativity, and improved overall survival for patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) who received inotuzumab ozogamicin (InO) vs standard of care chemotherapy (SC). Here we examined associations between genomic alterations and the efficacy of InO. Of 326 randomized patients, 91 (InO, n=43; SC, n=48) had samples evaluable for genomic analysis. The spectrum of gene fusions and other genomic alterations observed was comparable with prior studies of adult ALL. Responses to InO were observed in all leukemic subtypes, genomic alterations, and risk groups. Significantly higher rates of complete remission (CR)/CR with incomplete count recovery rates were observed with InO vs SC in patients with BCR::ABL1-like ALL (85.7% [6/7] vs 0% [0/5] P=0.0076), with TP53 alterations (100% [5/5] vs 12.5% [1/8], P=0.0047), and in the high-risk BCR::ABL1- (BCR::ABL1-like, low hypodiploid, KMT2A-rearranged) group (83.3% [10/12] vs 10.5% [2/19]; P<0.0001). This retrospective, exploratory analysis of the INO-VATE trial demonstrated potential for benefit with InO for patients with R/R ALL across leukemic subtypes, including BCR::ABL1-like ALL, and for those bearing diverse genomic alterations. Further confirmation of the efficacy of InO in patients with R/R ALL exhibiting the BCR::ABL1-like subtype or harboring TP53 alterations is warranted. This trial was registered at www.clinicaltrials.gov as no. NCT01564784.

Repression by RB1 characterizes genes involved in the penultimate stage of erythroid development.

Retinoblastoma-1 (RB1), and the RB1-related proteins p107 and p130, are key regulators of the cell cycle. Although RB1 is required for normal erythroid development in vitro, it is largely dispensable for erythropoiesis in vivo. The modest phenotype caused by RB1 deficiency in mice raises questions about redundancy within the RB1 family, and the role of RB1 in erythroid differentiation. Here we show that RB1 is the major pocket protein that regulates terminal erythroid differentiation. Erythroid cells lacking all pocket proteins exhibit the same cell cycle defects as those deficient for RB1 alone. RB1 has broad repressive effects on gene transcription in erythroid cells. As a group, RB1-repressed genes are generally well expressed but downregulated at the final stage of erythroid development. Repression correlates with E2F binding, implicating E2Fs in the recruitment of RB1 to repressed genes. Merging differential and time-dependent changes in expression, we define a group of approximately 800 RB1-repressed genes. Bioinformatics analysis shows that this list is enriched for terms related to the cell cycle, but also for terms related to terminal differentiation. Some of these have not been previously linked to RB1. These results expand the range of processes potentially regulated by RB1, and suggest that a principal role of RB1 in development is coordinating the events required for terminal differentiation.

A short linear motif in BNIP3L (NIX) mediates mitochondrial clearance in reticulocytes.

Elimination of defective mitochondria is essential for the health of long-lived, postmitotic cells. To gain insight into this process, we examined programmed mitochondrial clearance in reticulocytes. BNIP3L is a mitochondrial outer membrane protein that is required for clearance. It has been suggested that BNIP3L functions by causing mitochondrial depolarization, activating autophagy, or engaging the autophagy machinery. Here we showed in mice that BNIP3L activity localizes to a small region in its cytoplasmic domain, the minimal essential region (MER). The MER is a novel sequence, which comprises three contiguous hydrophobic amino acid residues, and flanking charged residues. Mutation of the central leucine residue causes complete loss of BNIP3L activity, and prevents rescue of mitochondrial clearance. Structural bioinformatics analysis predicts that the BNIP3L cytoplasmic domain lacks stable tertiary structure, but that the MER forms an α-helix upon binding to another protein. These findings support an adaptor model of BNIP3L, centered on the MER.

Mitochondrial clearance is regulated by Atg7-dependent and -independent mechanisms during reticulocyte maturation.

Mitochondrial clearance is a well recognized but poorly understood biologic process, and reticulocytes, which undergo programmed mitochondrial clearance, provide a useful model to study this phenomenon. At the ultrastructural level, mitochondrial clearance resembles an autophagy-related process; however, the role of autophagy in mitochondrial clearance has not been established. Here we provide genetic evidence that autophagy pathways, initially identified in yeast, are involved in mitochondrial clearance from reticulocytes. Atg7 is an autophagy protein and an E1-like enzyme, which is required for the activity of dual ubiquitin-like conjugation pathways. Atg7 is required for the conjugation of Atg12 to Atg5, and Atg8 to phosphatidylethanolamine (PE), and is essential for autophagosome formation. In the absence of Atg7, mitochondrial clearance from reticulocytes is diminished but not completely blocked. Mammalian homologs of Atg8 are unmodified in Atg7(-/-) erythroid cells, indicating that canonical autophagy pathways are inactive. Thus, mitochondrial clearance is regulated by both autophagy-dependent and -independent mechanisms. In addition, mitochondria, which depolarize in wild-type cells before elimination, remain polarized in Atg7(-/-) reticulocytes in culture. This suggests that mitochondrial depolarization is a consequence rather than a cause of autophagosome formation in reticulocytes.

NIX is required for programmed mitochondrial clearance during reticulocyte maturation.

The regulated clearance of mitochondria is a well recognized but poorly understood aspect of cellular homeostasis, and defects in this process have been linked to aging, degenerative diseases, and cancer. Mitochondria are recycled through an autophagy-related process, and reticulocytes, which completely eliminate their mitochondria during maturation, provide a physiological model to study this phenomenon. Here, we show that mitochondrial clearance in reticulocytes requires the BCL2-related protein NIX (BNIP3L). Mitochondrial clearance does not require BAX, BAK, BCL-X(L), BIM, or PUMA, indicating that NIX does not function through established proapoptotic pathways. Similarly, NIX is not required for the induction of autophagy during terminal erythroid differentiation. NIX is required for the selective elimination of mitochondria, however, because mitochondrial clearance, in the absence of NIX, is arrested at the stage of mitochondrial incorporation into autophagosomes and autophagosome maturation. These results yield insight into the mechanism of mitochondrial clearance in higher eukaryotes. Furthermore, they show a BAX- and BAK-independent role for a BCL2-related protein in development.

Role of erythropoietin receptor signaling in Friend virus-induced erythroblastosis and polycythemia.

Friend virus is an acutely oncogenic retrovirus that causes erythroblastosis and polycythemia in mice. Previous studies suggested that the Friend virus oncoprotein, gp55, constitutively activates the erythropoietin receptor (EPOR), causing uncontrolled erythroid proliferation. Those studies showed that gp55 confers growth factor independence on an interleukin-3 (IL-3)-dependent cell line (Ba/F3) when the EPOR is coexpressed. Subsequently, we showed that a truncated form of the stem-cell kinase receptor (sf-STK) is required for susceptibility to Friend disease. Given the requirement for sf-STK, we sought to establish the in vivo significance of gp55-mediated activation of the EPOR. We found that the cytoplasmic tyrosine residues of the EPOR, and signal transducer and activator of transcription-5 (STAT5), which acts through these sites, are not required for Friend virus-induced erythroblastosis. The EPOR itself was required for the development of erythroblastosis but not for gp55-mediated erythroid proliferation. Interestingly, the murine EPOR, which is required for gp55-mediated Ba/F3-cell proliferation, was dispensable for erythroblastosis in vivo. Finally, gp55-mediated activation of the EPOR and STAT5 are required for Friend virus-induced polycythemia. These results suggest that Friend virus activates both sf-STK and the EPOR to cause deregulated erythroid proliferation and differentiation.

Role of AP1/NFE2 binding sites in endogenous alpha-globin gene transcription.

High-level alpha-globin expression depends on cis-acting regulatory sequences located far upstream of the alpha-globin cluster. Sequences that contain the alpha-globin positive regulatory element (PRE) activate alpha-globin expression in transgenic mice. The alpha-globin PRE contains a pair of composite binding sites for the transcription factors activating protein 1 and nuclear factor erythroid 2 (AP1/NFE2). To determine the role of these binding sites in alpha-globin gene transcription, we mutated the AP1/NFE2 sites in the alpha-globin PRE in mice. We replaced the AP1/NFE2 sites with a neomycin resistance gene (neo) that is flanked by LoxP sites (floxed). Mice with this mutation exhibited increased embryonic death and alpha-thalassemia intermedia. Next, we removed the neo gene by Cre-mediated recombination, leaving a single LoxP site in place of the AP1/NFE2 sites. These mice were phenotypically normal. However, alpha-globin expression, measured by allele-specific RNA polymerase chain reaction (PCR), was decreased 25%. We examined the role of the hematopoietic-restricted transcription factor p45Nfe2 in activating expression through these sites and found that it is not required. Thus, we have demonstrated that AP1/NFE2 binding sites in the murine alpha-globin PRE contribute to long-range alpha-globin gene activation. The proteins that mediate this effect remain to be determined.