Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has only achieved success in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In this study we demonstrate the functional importance of autophagy in APL cell differentiation. We show that autophagy is increased during ATRA-induced granulocytic differentiation of the APL cell line NB4, and that this is associated with increased expression of LC3-II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin (sh)RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we show that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.

Retinoids are a family of signaling molecules derived from Vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

Objective In acute promyelocytic leukemia (APL), normal retinoid signaling is disrupted by an abnormal PML‐RARα fusion oncoprotein, leading to a block in cell differentiation. Therapeutic concentrations of all‐trans‐retinoic acid (ATRA) can restore retinoid‐induced transcription and promote degradation of the PML‐RARα protein. Autophagy is a catabolic pathway that utilizes lysosomal machinery to degrade intracellular material and facilitate cellular re‐modeling. Recent studies have identified autophagy as an integral component of ATRA‐induced myeloid differentiation. Methods As the molecular communication between retinoid signaling and the autophagy pathway is not defined, we performed RNA sequencing of NB4 APL cells treated with ATRA and examined autophagy‐related transcripts. Results ATRA altered the expression of >80 known autophagy‐related transcripts, including the key transcriptional regulator of autophagy and lysosomal biogenesis, TFEB (11.5‐fold increase). Induction of TFEB and its transcriptional target, sequestosome 1 (SQSTM1, p62), is reduced in ATRA‐resistant NB4R cells compared to NB4 cells. TFEB knockdown in NB4 cells alters the expression of transcriptional targets of TFEB and reduces CD11b transcript levels in response to ATRA. Conclusions We show for the first time that TFEB plays an important role in ATRA‐induced autophagy during myeloid differentiation and that autophagy induction potentiates leukemic cell differentiation (Note: this study includes data obtained from NCT00195156, https://clinicaltrials.gov/show/NCT00195156).

Ubiquitin/ISG15-conjugating E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post-translational protein modification that conjugates the ubiquitin-like modifier, interferon-stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all-trans-retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200-fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation-two cell line models were employed: the human APL cell line NB4 and its ATRA-resistant NB4R counterpart, as well as the ATRA-sensitive human AML HL60 cells along with their ATRA-resistant subclone-HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA-sensitive HL60 AML cells, but not in the ATRA-resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA-mediated UBE2L6 depletion in NB4 cells impeded ATRA-mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA-induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA-induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA-induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.Abbreviations AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ATRA, all-trans-retinoic acid; CML, chronic myeloid leukemia; HERC5, HECT and RLD domain containing E3 ubiquitin protein ligase 5; ISG15, interferon-stimulated gene 15; NBT, nitro blue tetrazolium; RARa, retinoic acid receptor alpha; UBE1L, ubiquitin-like modifier-activating enzyme 7; UBE2L6, ubiquitin/ISG15-conjugating enzyme E2L6; UBLs, ubiquitin-like modifiers; USP18, ubiquitin-specific peptidase 18.

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Background Seasonal influenza causes significant morbidity and mortality in allogeneic stem cell transplant (SCT) recipients. In this population, influenza virus can replicate for prolonged periods, despite neuraminidase inhibitor treatment, leading to resistance and treatment failure. Baloxavir targets the influenza polymerase and may be an effective treatment option in these patients. Methods We used baloxavir to treat five allogeneic SCT recipients that were still symptomatic and shedding influenza virus after completing one or more treatment courses of oseltamivir and characterized the viral isolates before and during treatment. Results Two patients were infected with influenza A/H1pdm09 carrying a neuraminidase variant (H275Y) linked to oseltamivir resistance. Both these two patients were successfully treated with baloxavir. Of the three patients infected with wild‐type influenza virus, two cleared the virus after baloxavir treatment, while the third patient developed the polymerase I38T variant linked to baloxavir resistance. Conclusions Our data suggest that baloxavir treatment can be effective in treating neuraminidase inhibitor‐resistant influenza in profoundly immunocompromised patients. Randomized clinical trials are needed to define the role of baloxavir alone and combined with oseltamivir for the treatment of influenza in SCT recipients and other immunocompromised populations.

We conducted a prospective evaluation of cord blood (CB)–derived adoptive cell therapy, after salvage chemotherapy, for patients with advanced myeloid malignancies and poor prognosis. Previously, we reported safety, feasibility, and preliminary efficacy of this approach. We present updated results in 31 patients who received intensive chemotherapy followed by CB infusion and identify predictors of response. To enhance the antileukemic effect, we selected CB units (CBU) with shared inherited paternal antigens and/or noninherited maternal antigens with the recipients. Twenty-eight patients with acute myeloid leukemia (AML), 2 with myelodysplastic syndrome, and 1 in chronic myeloid leukemia myeloid blast crisis were enrolled; 9 had relapsed after allogeneic transplant. Response was defined as <5% blasts in hypocellular bone marrow at 2 weeks after treatment. Thirteen patients (42%) responded; a rate higher than historical data with chemotherapy only. Twelve had CBU-derived chimerism detected; chimerism was a powerful predictor of response (P < .001). CBU lymphocyte content and a prior transplant were associated with chimerism (P < .01). Safety was acceptable: 3 patients developed mild cytokine release syndrome, 2 had grade 1 and 2 had grade 4 graft-versus-host disease. Seven responders and 6 nonresponders (after additional therapy) received subsequent transplant; 5 are alive (follow-up, 5-47 months). The most common cause of death for nonresponders was disease progression, whereas for responders it was infection. CB-derived adoptive cell therapy is feasible and efficacious for refractory AML. Banked CBU are readily available for treatment. Response depends on chimerism, highlighting the graft-versus-leukemia effect of CB cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT02508324.

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