HSPA8 chaperone complex drives chaperone-mediated autophagy regulation in acute promyelocytic leukemia cell differentiation.

INTRODUCTION: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused towards tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML has remains elusive. METHODS: We took advantages of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally, but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cAMP. RESULTS: Here, we report that CMA related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA resistant NB4-R1 cells to differentiate upon ATRA treatment, but reduced association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), which are necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. CONCLUSION: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.

Unveiling CXCR2 as a promising therapeutic target in renal cell carcinoma: exploring the immunotherapeutic paradigm shift through its inhibition by RCT001.

BACKGROUND: In clear cell renal cell carcinoma (ccRCC), first-line treatment combines nivolumab (anti-PD-1) and ipilimumab (anti-CTLA4), yielding long-term remissions but with only a 40% success rate. Our study explored the potential of enhancing ccRCC treatment by concurrently using CXCR2 inhibitors alongside immunotherapies. METHODS: We analyzed ELR + CXCL levels and their correlation with patient survival during immunotherapy. RCT001, a unique CXCR2 inhibitor, was examined for its mechanism of action, particularly its effects on human primary macrophages. We tested the synergistic impact of RCT001 in combination with immunotherapies in both mouse models of ccRCC and human ccRCC in the presence of human PBMC. RESUTS: Elevated ELR + CXCL cytokine levels were found to correlate with reduced overall survival during immunotherapy. RCT001, our optimized compound, acted as an inverse agonist, effectively inhibiting angiogenesis and reducing viability of primary ccRCC cells. It redirected M2-like macrophages without affecting M1-like macrophage polarization directed against the tumor. In mouse models, RCT001 enhanced the efficacy of anti-CTLA4 + anti-PD1 by inhibiting tumor-associated M2 macrophages and tumor-associated neutrophils. It also impacted the activation of CD4 T lymphocytes, reducing immune-tolerant lymphocytes while increasing activated natural killer and dendritic cells. Similar effectiveness was observed in human RCC tumors when RCT001 was combined with anti-PD-1 treatment. CONCLUSIONS: RCT001, by inhibiting CXCR2 through its unique mechanism, effectively suppresses ccRCC cell proliferation, angiogenesis, and M2 macrophage polarization. This optimization potentiates the efficacy of immunotherapy and holds promise for significantly improving the survival prospects of metastatic ccRCC patients.

The Antioxidant TEMPOL Protects Human Hematopoietic Stem Cells From Culture-Mediated Loss of Functions.

In a steady state, hematopoietic stem cells (HSC) exhibit very low levels of reactive oxygen species (ROS). Upon stress, HSC get activated and enter into proliferation and differentiation process to ensure blood cell regeneration. Once activated, their levels of ROS increase, as messengers to mediate their proliferation and differentiation programs. However, at the end of the stress episode, ROS levels need to return to normal to avoid HSC exhaustion. It was shown that antioxidants can prevent loss of HSC self-renewal potential in several contexts such as aging or after exposure to low doses of irradiation suggesting that antioxidants can be used to maintain HSC functional properties upon culture-induced stress. Indeed, in humans, HSC are increasingly used for cell and gene therapy approaches, requiring them to be cultured for several days. As expected, we show that a short culture period leads to drastic defects in HSC functional properties. Moreover, a switch of HSC transcriptional program from stemness to differentiation was evidenced in cultured HSC. Interestingly, cultured-HSC treated with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO or Tempol) exhibited a higher clonogenic potential in secondary colony forming unit cell (CFU-C) assay and higher reconstitution potential in xenograft model, compared to untreated cultured-HSC. By transcriptomic analyses combined with serial CFU-C assays, we show that Tempol, which mimics superoxide dismutase, protects HSC from culture-induced stress partly through VEGFα signaling. Thus, we demonstrate that adding Tempol leads to the protection of HSC functional properties during ex vivo culture.

The generation, activation, and polarization of monocyte-derived macrophages in human malignancies.

Macrophages are immune cells that originate from embryogenesis or from the differentiation of monocytes. They can adopt numerous phenotypes depending on their origin, tissue distribution and in response to different stimuli and tissue environment. Thus, in vivo, macrophages are endowed with a continuum of phenotypes that are rarely strictly pro-inflammatory or anti-inflammatory and exhibit a broad expression profile that sweeps over the whole polarization spectrum. Schematically, three main macrophage subpopulations coexist in human tissues: naïve macrophages also called M0, pro-inflammatory macrophages referred as M1 macrophages, and anti-inflammatory macrophages also known as M2 macrophages. Naïve macrophages display phagocytic functions, recognize pathogenic agents, and rapidly undergo polarization towards pro or anti-inflammatory macrophages to acquire their full panel of functions. Pro-inflammatory macrophages are widely involved in inflammatory response, during which they exert anti-microbial and anti-tumoral functions. By contrast, anti-inflammatory macrophages are implicated in the resolution of inflammation, the phagocytosis of cell debris and tissue reparation following injuries. Macrophages also play important deleterious or beneficial roles in the initiation and progression of different pathophysiological settings including solid and hematopoietic cancers. A better understanding of the molecular mechanisms involved in the generation, activation and polarization of macrophages is a prerequisite for the development of new therapeutic strategies to modulate macrophages functions in pathological situations.

Caspase Inhibition Modulates Monocyte-Derived Macrophage Polarization in Damaged Tissues.

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues.

MCB-613 exploits a collateral sensitivity in drug resistant EGFR-mutant non-small cell lung cancer through covalent inhibition of KEAP1.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR-mutant NSCLC cells.

High caspase 3 and vulnerability to dual BCL2 family inhibition define ETO2::GLIS2 pediatric leukemia.

Pediatric acute myeloid leukemia expressing the ETO2::GLIS2 fusion oncogene is associated with dismal prognosis. Previous studies have shown that ETO2::GLIS2 can efficiently induce leukemia development associated with strong transcriptional changes but those amenable to pharmacological targeting remained to be identified. By studying an inducible ETO2::GLIS2 cellular model, we uncovered that de novo ETO2::GLIS2 expression in human cells led to increased CASP3 transcription, CASP3 activation, and cell death. Patient-derived ETO2::GLIS2+ leukemic cells expressed both high CASP3 and high BCL2. While BCL2 inhibition partly inhibited ETO2::GLIS2+ leukemic cell proliferation, BH3 profiling revealed that it also sensitized these cells to MCL1 inhibition indicating a functional redundancy between BCL2 and MCL1. We further show that combined inhibition of BCL2 and MCL1 is mandatory to abrogate disease progression using in vivo patient-derived xenograft models. These data reveal that a transcriptional consequence of ETO2::GLIS2 expression includes a positive regulation of the pro-apoptotic CASP3 and associates with a vulnerability to combined targeting of two BCL2 family members providing a novel therapeutic perspective for this aggressive pediatric AML subgroup.

Real-life challenges using personalized prognostic scoring systems in acute myeloid leukemia.

Personalized medicine is a challenge for patients with acute myeloid leukemia (AML). The identification of several genetic mutations in several AML trials led to the creation of a personalized prognostic scoring algorithm known as the Knowledge Bank (KB). In this study, we assessed the prognostic value of this algorithm on a cohort of 167 real life AML patients. We compared KB predicted outcomes to real-life outcomes. For patients younger than 60-year-old, OS was similar in favorable and intermediate ELN risk category. However, KB algorithm failed to predict OS for younger patients in the adverse ELN risk category and for patients older than 60 years old in the favorable ELN risk category. These discrepancies may be explained by the emergence of several new therapeutic options as well as the improvement of allogeneic stem cell transplantation (aHSCT) outcomes and supportive cares. Personalized medicine is a major challenge and predictions models are powerful tools to predict patient's outcome. However, the addition of new therapeutic options in the field of AML requires a prospective validation of these scoring systems to include recent therapeutic innovations.

BTG1 inactivation drives lymphomagenesis and promotes lymphoma dissemination through activation of BCAR1.

Understanding the functional role of mutated genes in cancer is required to translate the findings of cancer genomics into therapeutic improvement. BTG1 is recurrently mutated in the MCD/C5 subtype of diffuse large B-cell lymphoma (DLBCL), which is associated with extranodal dissemination. Here, we provide evidence that Btg1 knock out accelerates the development of a lethal lymphoproliferative disease driven by Bcl2 overexpression. Furthermore, we show that the scaffolding protein BCAR1 is a BTG1 partner. Moreover, after BTG1 deletion or expression of BTG1 mutations observed in patients with DLBCL, the overactivation of the BCAR1-RAC1 pathway confers increased migration ability in vitro and in vivo. These modifications are targetable with the SRC inhibitor dasatinib, which opens novel therapeutic opportunities in BTG1 mutated DLBCL.

P2RY2-AKT activation is a therapeutically actionable consequence of XPO1 inhibition in acute myeloid leukemia.

Selinexor is a first-in-class inhibitor of the nuclear exportin XPO1 that was recently approved by the US Food and Drug Administration for the treatment of multiple myeloma and diffuse large B-cell lymphoma. In relapsed/refractory acute myeloid leukemia (AML), selinexor has shown promising activity, suggesting that selinexor-based combination therapies may have clinical potential. Here, motivated by the hypothesis that selinexor's nuclear sequestration of diverse substrates imposes pleiotropic fitness effects on AML cells, we systematically catalog the pro- and anti-fitness consequences of selinexor treatment. We discover that selinexor activates PI3Kγ-dependent AKT signaling in AML by upregulating the purinergic receptor P2RY2. Inhibiting this axis potentiates the anti-leukemic effects of selinexor in AML cell lines, patient-derived primary cultures and multiple mouse models of AML. In a syngeneic, MLL-AF9-driven mouse model of AML, treatment with selinexor and ipatasertib outperforms both standard-of-care chemotherapy and chemotherapy with selinexor. Together, these findings establish drug-induced P2RY2-AKT signaling as an actionable consequence of XPO1 inhibition in AML.

Reprogramming monocyte-derived macrophages through caspase inhibition.

Macrophages are widely distributed innate immune cells that play an indispensable role in a variety of physiologic and pathologic processes, including organ development, host defense, acute and chronic inflammation, solid and hematopoietic cancers. Beyond their inextricable role as conveyors of programmed cell death, we have previously highlighted that caspases exert non-apoptotic functions, especially during the differentiation of monocyte-derived cells in response to CSF-1. Here, we found that non-canonic cleavages of caspases, reflecting their activation, are maintained during IL-4-induced monocyte-derived macrophages polarization. Moreover, Emricasan, a pan-caspase inhibitor that demonstrated promising preclinical activity in various diseases and safely entered clinical testing for the treatment of liver failure, prevents the generation and the anti-inflammatory polarization of monocyte-derived macrophages ex vivo. Interestingly, caspase inhibition also triggered the reprogramming of monocyte-derived cells evidenced by RNA sequencing. Taken together, our findings position Emricasan as a potential alternative to current therapies for reprogramming macrophages in diseases driven by monocyte-derived macrophages.

AMPK-PERK axis represses oxidative metabolism and enhances apoptotic priming of mitochondria in acute myeloid leukemia.

AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.

Ultrasound-assisted one-pot three-component synthesis of new isoxazolines bearing sulfonamides and their evaluation against hematological malignancies.

In the present study, following a one-pot two-step protocol, we have synthesized novel sulfonamides-isoxazolines hybrids (3a-r) via a highly regioselective 1,3-dipolar cycloaddition. The present methodology capitalized on trichloroisocyanuric acid (TCCA) as a safe and ecological oxidant and chlorinating agent for the in-situ conversion of aldehydes to nitrile oxides in the presence of hydroxylamine hydrochloride, under ultrasound activation. These nitrile oxides could be engaged in 1,3-dipolar cycloaddition reactions with various alkene to afford the targeted sulfonamides-isoxazolines hybrids (3a-r). The latter were assessed for their antineoplastic activity against model leukemia cell lines (Chronic Myeloid Leukemia, K562 and Promyelocytic Leukemia, HL-60).

Dual Covalent Inhibition of PKM and IMPDH Targets Metabolism in Cutaneous Metastatic Melanoma.

Overcoming acquired drug resistance is a primary challenge in cancer treatment. Notably, more than 50% of patients with BRAFV600E cutaneous metastatic melanoma (CMM) eventually develop resistance to BRAF inhibitors. Resistant cells undergo metabolic reprogramming that profoundly influences therapeutic response and promotes tumor progression. Uncovering metabolic vulnerabilities could help suppress CMM tumor growth and overcome drug resistance. Here we identified a drug, HA344, that concomitantly targets two distinct metabolic hubs in cancer cells. HA344 inhibited the final and rate-limiting step of glycolysis through its covalent binding to the pyruvate kinase M2 (PKM2) enzyme, and it concurrently blocked the activity of inosine monophosphate dehydrogenase, the rate-limiting enzyme of de novo guanylate synthesis. As a consequence, HA344 efficiently targeted vemurafenib-sensitive and vemurafenib-resistant CMM cells and impaired CMM xenograft tumor growth in mice. In addition, HA344 acted synergistically with BRAF inhibitors on CMM cell lines in vitro. Thus, the mechanism of action of HA344 provides potential therapeutic avenues for patients with CMM and a broad range of different cancers. SIGNIFICANCE: Glycolytic and purine synthesis pathways are often deregulated in therapy-resistant tumors and can be targeted by the covalent inhibitor described in this study, suggesting its broad application for overcoming resistance in cancer.

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

Plk1, upregulated by HIF-2, mediates metastasis and drug resistance of clear cell renal cell carcinoma.

Polo-like kinase 1 (Plk1) expression is inversely correlated with survival advantages in many cancers. However, molecular mechanisms that underlie Plk1 expression are poorly understood. Here, we uncover a hypoxia-regulated mechanism of Plk1-mediated cancer metastasis and drug resistance. We demonstrated that a HIF-2-dependent regulatory pathway drives Plk1 expression in clear cell renal cell carcinoma (ccRCC). Mechanistically, HIF-2 transcriptionally targets the hypoxia response element of the Plk1 promoter. In ccRCC patients, high expression of Plk1 was correlated to poor disease-free survival and overall survival. Loss-of-function of Plk1 in vivo markedly attenuated ccRCC growth and metastasis. High Plk1 expression conferred a resistant phenotype of ccRCC to targeted therapeutics such as sunitinib, in vitro, in vivo, and in metastatic ccRCC patients. Importantly, high Plk1 expression was defined in a subpopulation of ccRCC patients that are refractory to current therapies. Hence, we propose a therapeutic paradigm for improving outcomes of ccRCC patients.

Real-life experience with CPX-351 and impact on the outcome of high-risk AML patients: a multicentric French cohort.

CPX-351 is a liposomal formulation of cytarabine and daunorubicin approved for the treatment of adults with newly diagnosed, therapy-related acute myeloid leukemia (t-AML) or AML with myelodysplasia-related changes (MRC-AML). We retrospectively analyzed the efficacy and safety of CPX-351 in a real-world setting in 103 patients from 12 French centers, including the evaluation of molecular abnormalities at baseline and minimal residual disease (MRD) in responding patients, compared with a historical data set from Bordeaux-Toulouse DATAML registry. A favorable safety profile was observed, with a low frequency of alopecia (11%) and gastrointestinal toxicity (50%). The overall response rate after induction was 59%, and MRD <10-3 was achieved in 57% of complete response (CR)/CR with incomplete hematological recovery (CRi) patients. Only the presence of mutated TP53 (P = .02) or PTPN11 (P = .004) predicted lower response in multivariate analysis. Interestingly, high-risk molecular prognosis subgroups defined by 2017 European LeukemiaNet risk stratification, including ASXL1 and RUNX1 mutations, were not associated with a significantly lower response rate using CPX-351. With a median follow-up of 8.6 months, median overall survival (OS) was 16.1 months. Thirty-six patients underwent allogeneic stem cell transplantation with a significantly longer median OS compared with nontransplanted patients (P < .001). In multivariate analyses, only spliceosome mutations were associated with better OS (P = .04). In comparison with intensive chemotherapy, there was no difference in OS for patients <60 years. These data confirm the efficacy and safety of CPX-351 in high-risk AML (t-AML and MRC-AML) in a real-life setting. CPX-351 is a treatment of choice for patients aged ≥60 years.

Drug Resistance in Hematological Malignancies.

Hematological malignancies define a highly heterogeneous set of blood-, bone marrow-, and organ-associated diseases with highly variable prognoses that constantly relapse upon treatment [...].

Retraction Note: LAMP2 expression dictates azacytidine response and prognosis in MDS/AML.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.