Acquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

Loss of Dynamin 2 GTPase function results in microcytic anaemia.

In a dominant mouse ethylnitrosurea mutagenesis screen for genes regulating erythropoiesis, we identified a pedigree with a novel microcytic hypochromia caused by a V235G missense mutation in Dynamin 2 (Dnm2). Mutations in Dnm2, a GTPase, are highly disease-specific and have been implicated in four forms of human diseases: centronuclear myopathy, Charcot-Marie Tooth neuropathy and, more recently, T-cell leukaemia and Hereditary Spastic Paraplegia, but red cell abnormalities have not been reported to date. The V235G mutation lies within a crucial GTP nucleotide-binding pocket of Dnm2, and resulted in defective GTPase activity and incompatibility with life in the homozygous state. Dnm2 is an essential mediator of clathrin-mediated endocytosis, which is required for the uptake of transferrin (Tf) into red cells for incorporation of haem. Accordingly, we observed significantly reduced Tf uptake by Dnm2+/V235G cells, which led to impaired endosome formation. Despite these deficiencies, surprisingly all iron studies were unchanged, suggesting an unexplained alternative mechanism underlies microcytic anaemia in Dnm2+/V235G mice. This study provides the first in vivo evidence for the requirements of Dnm2 in normal erythropoiesis.

Genomics of primary chemoresistance and remission induction failure in paediatric and adult acute myeloid leukaemia.

Cure rates of children and adults with acute myeloid leukaemia (AML) remain unsatisfactory partly due to chemotherapy resistance. We investigated the genetic basis of AML in 107 primary cases by sequencing 670 genes mutated in haematological malignancies. SETBP1, ASXL1 and RELN mutations were significantly associated with primary chemoresistance. We identified genomic alterations not previously described in AML, together with distinct genes that were significantly overexpressed in therapy-resistant AML. Defined gene mutations were sufficient to explain primary induction failure in only a minority of cases. Thus, additional genetic or molecular mechanisms must cause primary chemoresistance in paediatric and adult AML.

Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model.

We used an N-ethyl-N-nitrosurea-based forward genetic screen in mice to identify new genes and alleles that regulate erythropoiesis. Here, we describe a mouse line expressing an activated form of the K-Cl cotransporter Slc12a4 (Kcc1), which results in a semi-dominant microcytosis of red cells. A missense mutation from methionine to lysine in the cytoplasmic tail of Kcc1 impairs phosphorylation of adjacent threonines required for inhibiting cotransporter activity. We bred Kcc1(M935K) mutant mice with a humanized mouse model of sickle cell disease to directly explore the relevance of the reported increase in KCC activity in disease pathogenesis. We show that a single mutant allele of Kcc1 induces widespread sickling and tissue damage, leading to premature death. This mouse model reveals important new insights into the regulation of K-Cl cotransporters and provides in vivo evidence that increased KCC activity worsened end-organ damage and diminished survival in sickle cell disease.

Patient-Derived Xenograft Models for Leukemias.

Patient-derived xenograft (PDX) modeling is a valuable tool for the study of leukemia pathogenesis, progression, and therapy response. Engraftment of human leukemia cells occurs following injection into the tail vein (or retro-orbital vein) of preconditioned immunocompromised mice. Injected mice are maintained in a sterile and supportive housing environment until leukemia engraftment is observed, at which time studies such as drug treatments or leukemia sampling can occur. Here, we outline a method for generating PDXs from Acute Myeloid Leukemia (AML) patient samples using tail vein injection; however it can also be readily applied to T- and B- Acute Lymphoblastic Leukemia (ALL) samples.

Putting the STING back into BH3-mimetic drugs for TP53-mutant blood cancers.

TP53-mutant blood cancers remain a clinical challenge. BH3-mimetic drugs inhibit BCL-2 pro-survival proteins, inducing cancer cell apoptosis. Despite acting downstream of p53, functional p53 is required for maximal cancer cell killing by BH3-mimetics through an unknown mechanism. Here, we report p53 is activated following BH3-mimetic induced mitochondrial outer membrane permeabilization, leading to BH3-only protein induction and thereby potentiating the pro-apoptotic signal. TP53-deficient lymphomas lack this feedforward loop, providing opportunities for survival and disease relapse after BH3-mimetic treatment. The therapeutic barrier imposed by defects in TP53 can be overcome by direct activation of the cGAS/STING pathway, which promotes apoptosis of blood cancer cells through p53-independent BH3-only protein upregulation. Combining clinically relevant STING agonists with BH3-mimetic drugs efficiently kills TRP53/TP53-mutant mouse B lymphoma, human NK/T lymphoma, and acute myeloid leukemia cells. This represents a promising therapy regime that can be fast-tracked to tackle TP53-mutant blood cancers in the clinic.

Acquired BCL2 variants associated with venetoclax resistance in acute myeloid leukemia.

We report and characterize three venetoclax-resistant BCL2 variants arising during venetoclax/azacitidine therapy in acute myeloid leukemia (AML). Our results indicate the potential for on-target venetoclax resistance in patients with AML at relapse.

ENU mutagenesis identifies the first mouse mutants reproducing human ß-thalassemia at the genomic level.

Forward genetic screens have been performed in many species to identify phenotypes in specific organ systems. We have undertaken a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify dominant mutations that perturb erythropoiesis in mice. Mutant mice that displayed an erythrocyte mean cell volume (MCV) greater than three standard deviations from the population mean were identified. Two of these lines, RBC13 and RBC14, displayed a hypochromic, microcytic anemia, accompanied by a marked reticulocytosis, splenomegaly and diminished red cell survival. Timed pregnancies from heterozygous intercrosses revealed that a quarter of the embryos displayed severe anemia and did not survive beyond embryonic day (E) 18.5, consistent with homozygous ß-thalassemia. Genetic complementation studies with a ß-thalassemia mouse line reproduced the embryonic lethality in compound heterozygotes and a genomic custom capture array and massively parallel sequencing of the ß-globin locus identified the causative mutations. The RBC13 line displayed a nonsense mutation at codon 40 in exon 2 of the ß-major gene, invoking parallels with the common ß(0)39 thalassemia mutation seen in humans. The RBC14 line exhibited a mutation at the polyadenylation signal of the ß-major gene, exactly replicating a human ß-thalassemia mutation. The RBC13 and RBC14 lines are the first ß-thalassemia mouse models that reproduce human ß-thalassemia at the genomic level, and as such highlight the power of ENU mutagenesis screens in generating mouse models of human disease.

Cellular distribution of IDH mutations in AML during morphologic remission.

Limited information exists about the cellular distribution of mutations which persist in remission in acute myeloid leukemia (AML) (variably considered pre-leukemic mutations). We hypothesized that mutations detectable in all cell compartments may be less pathogenic than those that are myeloid-restricted. Here, we describe the cellular compartments that have IDH mutations in five patients with IDH-mutated AML in morphologic remission. Unlike pre-leukemic clones harboring the more common DNMT3A, TET2 and ASXL1 (DTA) mutations, we show that IDH mutations are myeloid-restricted. This finding provides an explanation for the reports that IDH mutations carry a higher risk for relapse than DTA mutations. Detailed analysis of one case also shows acquisition of additional mutations in distinct cellular compartments, illustrating subclonal complexity associated with therapeutics.

Inhibition of pyrimidine biosynthesis targets protein translation in acute myeloid leukemia.

The mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins, and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anticancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein, we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) and has potent and selective activity against multiple AML subtypes. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

Author Correction: KCC1 Activation protects Mice from the Development of Experimental Cerebral Malaria.

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

Chemotherapy and Venetoclax in Elderly Acute Myeloid Leukemia Trial (CAVEAT): A Phase Ib Dose-Escalation Study of Venetoclax Combined With Modified Intensive Chemotherapy.

PURPOSE: The B-cell lymphoma 2 (BCL-2) inhibitor venetoclax has an emerging role in acute myeloid leukemia (AML), with promising response rates in combination with hypomethylating agents or low-dose cytarabine in older patients. The tolerability and efficacy of venetoclax in combination with intensive chemotherapy in AML is unknown. PATIENTS AND METHODS: Patients with AML who were ≥ 65 years (≥ 60 years if monosomal karyotype) and fit for intensive chemotherapy were allocated to venetoclax dose-escalation cohorts (range, 50-600 mg). Venetoclax was administered orally for 14 days each cycle. During induction, a 7-day prephase/dose ramp-up (days -6 to 0) was followed by an additional 7 days of venetoclax combined with infusional cytarabine 100 mg/m2 on days 1-5 and idarubicin 12 mg/m2 intravenously on days 2-3 (ie, 5 + 2). Consolidation (4 cycles) included 14 days of venetoclax (days -6 to 7) combined with cytarabine (days 1-2) and idarubicin (day 1). Maintenance venetoclax was permitted (7 cycles). The primary objective was to assess the optimal dose schedule of venetoclax with 5 + 2. RESULTS: Fifty-one patients with a median age of 72 years (range, 63-80 years) were included. The maximum tolerated dose was not reached with venetoclax 600 mg/day. The main grade ≥ 3 nonhematologic toxicities during induction were febrile neutropenia (55%) and sepsis (35%). In contrast to induction, platelet recovery was notably delayed during consolidation cycles. The overall response rate (complete remission [CR]/CR with incomplete count recovery) was 72%; it was 97% in de novo AML and was 43% in secondary AML. During the venetoclax prephase, marrow blast reductions (≥ 50%) were noted in NPM1-, IDH2-, and SRSF2-mutant AML. CONCLUSION: Venetoclax combined with 5 + 2 induction chemotherapy was safe and tolerable in fit older patients with AML. Although the optimal postremission therapy remains to be determined, the high remission rate in de novo AML warrants additional investigation (ANZ Clinical Trial Registry No. ACTRN12616000445471).

KCC1 Activation protects Mice from the Development of Experimental Cerebral Malaria.

Plasmodium falciparum malaria causes half a million deaths per year, with up to 9% of this mortality caused by cerebral malaria (CM). One of the major processes contributing to the development of CM is an excess of host inflammatory cytokines. Recently K+ signaling has emerged as an important mediator of the inflammatory response to infection; we therefore investigated whether mice carrying an ENU induced activation of the electroneutral K+ channel KCC1 had an altered response to Plasmodium berghei. Here we show that Kcc1M935K/M935K mice are protected from the development of experimental cerebral malaria, and that this protection is associated with an increased CD4+ and TNFa response. This is the first description of a K+ channel affecting the development of experimental cerebral malaria.

Bone marrow transplantation corrects haemolytic anaemia in a novel ENU mutagenesis mouse model of TPI deficiency.

In this study, we performed a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice to identify novel genes or alleles that regulate erythropoiesis. Here, we describe a recessive mouse strain, called RBC19, harbouring a point mutation within the housekeeping gene, Tpi1, which encodes the glycolysis enzyme, triosephosphate isomerase (TPI). A serine in place of a phenylalanine at amino acid 57 severely diminishes enzyme activity in red blood cells and other tissues, resulting in a macrocytic haemolytic phenotype in homozygous mice, which closely resembles human TPI deficiency. A rescue study was performed using bone marrow transplantation of wild-type donor cells, which restored all haematological parameters and increased red blood cell enzyme function to wild-type levels after 7 weeks. This is the first study performed in a mammalian model of TPI deficiency, demonstrating that the haematological phenotype can be rescued.

Characterization of Tfrc-mutant mice with microcytic phenotypes.

To identify novel regulators of erythropoiesis, we performed independent forward genetic screens using the chemical mutagen ENU in mice. Among progeny displaying microcytic red-cell phenotypes, 7 independent mouse strains harboring mutations within the transferrin receptor gene Tfrc were identified. Six of the mutants, including the previously described red blood cell 6 (RBC6) strain, displayed reduced erythroblast CD71 expression and midgestation lethality of homozygotes (E12.5-E14.5), and 1 novel strain, RBC21, displayed a variable phenotype with sustained CD71 expression and late homozygous lethality (E18.5). Standard iron studies were normal in the RBC21 mutant, but intracellular ferritin was significantly reduced. The microcytic phenotype seen in the RBC21 strain was the result of impaired binding of transferrin to the receptor. Neither RBC6 nor RBC21 responded to iron replacement therapy. These studies describe how point mutations of the transferrin receptor can cause a microcytic anemia that does not respond to iron therapy and would not be detected by routine iron studies, such as serum ferritin.

DPP8/DPP9 inhibitor-induced pyroptosis for treatment of acute myeloid leukemia.

Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death3, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.