FBXO21 mediated degradation of p85α regulates proliferation and survival of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of myeloid blasts in the bone marrow (BM). Despite advances in therapy, the prognosis for AML patients remains poor, and there is a need to identify novel molecular pathways regulating tumor cell survival and proliferation. F-box ubiquitin E3 ligase, FBXO21, has low expression in AML, but expression correlates with survival in AML patients and patients with higher expression have poorer outcomes. Silencing FBXO21 in human-derived AML cell lines and primary patient samples leads to differentiation, inhibition of tumor progression, and sensitization to chemotherapy agents. Additionally, knockdown of FBXO21 leads to up-regulation of cytokine signaling pathways. Through a mass spectrometry-based proteomic analysis of FBXO21 in AML, we identified that FBXO21 ubiquitylates p85α, a regulatory subunit of the phosphoinositide 3-kinase (PI3K) pathway, for degradation resulting in decreased PI3K signaling, dimerization of free p85α and ERK activation. These findings reveal the ubiquitin E3 ligase, FBXO21, plays a critical role in regulating AML pathogenesis, specifically through alterations in PI3K via regulation of p85α protein stability.

Synthetic Immunotherapy: Programming Immune Cells with Novel and Sophisticated Logic Capabilities.

The development of chimeric antigen receptor (CAR) T cells began as a means toward specific yet modular therapies against cancer. Recent advancements in several CAR T cell therapies show the promise of cellular immunotherapy in cancer treatment. CAR T cell therapy is still immature, however, and improvements are needed to fully realize its curative potential. The approved CAR T cells are designed with simple logic capabilities; an antigen sensor that, when bound to the target antigen, triggers costimulation domains and native T cell activation. This single-type sensor and native activation design, although capable, also has severe limitations. Reliance on a single-type sensor leads to unwanted toxicity toward antigen-expressing normal tissues, and unmodulated activation leads to unwanted cytokine toxicity. Synthetic biology (SB) offers a powerful solution to these limitations: modular receptors with customizable sensors and output behaviors that enable higher Boolean logic. SB T cells already have shown incredible capabilities, such as multiple-antigen discrimination and improved persistence. In light of these results, cellular immunotherapy may already be branching into a new subfield that we term here as "synthetic immunotherapy." Here we review the current logic capabilities of CAR T cells, the resulting limitations, and the engineering undertaken to address these issues. We then discuss several tools of SB and show how SB CAR T cells pave the way for synthetic immunotherapy.

Integrating geriatric assessment and genetic profiling to personalize therapy selection in older adults with acute myeloid leukemia.

INTRODUCTION: Survival benefit associated with intensive over low-intensity chemotherapy in older adults with acute myeloid leukemia (AML) is controversial. Geriatric assessment and genetic risk categories correlate with survival following intensive chemotherapy in older adults with AML and can guide treatment selection. MATERIALS AND METHODS: In a single-center trial, we integrated both geriatric assessment, and genetic risk categories to personalize selection of intensive versus low-intensity chemotherapy in older adults ≥60 years with AML (NCT03226418). In the present report, we demonstrate feasibility of this approach. RESULTS: Broad eligibility criteria and co-management of patients with community oncologists allowed enrollment of 45% of all patients with AML treated at our center during the study period. The median time from enrollment to therapy initiation was two days (range 0-9). Over half of the trial patients had a score of ≥3 on hematopoietic cell transplantation comorbidity index, impairment in physical function (Short Physical Performance Battery), and Montreal Cognitive Assessment. Three fit patients received intensive chemotherapy, whereas other patients received low-intensity chemotherapy. Mortality at 30 days from diagnosis was 3.7% (95% confidence interval [CI] 0.7-18.3%) and at 90 days was 29.6% (95% CI 15.9-48.5%). One-year overall survival was 66% (95% CI 60-87%). DISCUSSION: Our data demonstrate the feasibility of integrating geriatric assessment in precision oncology trials to define fitness for intensive chemotherapy. Broad eligibility criteria and academic-community collaboration can expand access to clinical trials.

Multi-omics reveals mitochondrial metabolism proteins susceptible for drug discovery in AML.

Acute myeloid leukemia (AML) is a devastating cancer affecting the hematopoietic system. Previous research has relied on RNA sequencing and microarray techniques to study the downstream effects of genomic alterations. While these studies have proven efficacious, they fail to capture the changes that occur at the proteomic level. To interrogate the effect of protein expression alterations in AML, we performed a quantitative mass spectrometry in parallel with RNAseq analysis using AML mouse models. These combined results identified 34 proteins whose expression was upregulated in AML tumors, but strikingly, were unaltered at the transcriptional level. Here we focus on mitochondrial electron transfer proteins ETFA and ETFB. Silencing of ETFA and ETFB led to increased mitochondrial activity, mitochondrial stress, and apoptosis in AML cells, but had little to no effect on normal human CD34+ cells. These studies identify a set of proteins that have not previously been associated with leukemia and may ultimately serve as potential targets for therapeutic manipulation to hinder AML progression and help contribute to our understanding of the disease.

RUNX1 and CBFß-SMMHC transactivate target genes together in abnormal myeloid progenitors for leukemia development.

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. It is generally considered that CBFß-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1. However, recent findings challenge the RUNX1-repression model for CBFß-SMMHC-mediated leukemogenesis. To definitively address the role of Runx1 in CBFB-MYH11-induced leukemia, we crossed conditional Runx1 knockout mice (Runx1f/f) with conditional Cbfb-MYH11 knockin mice (Cbfb+/56M). On Mx1-Cre activation in hematopoietic cells induced by poly (I:C) injection, all Mx1-CreCbfb+/56M mice developed leukemia in 5 months, whereas no leukemia developed in Runx1f/fMx1-CreCbfb+/56M mice, and this effect was cell autonomous. Importantly, the abnormal myeloid progenitors (AMPs), a leukemia-initiating cell population induced by Cbfb-MYH11 in the bone marrow, decreased and disappeared in Runx1f/fMx1-CreCbfb+/56M mice. RNA-seq analysis of AMP cells showed that genes associated with proliferation, differentiation blockage, and leukemia initiation were differentially expressed between Mx1-CreCbfb+/56M and Runx1f/fMx1-CreCbfb+/56M mice. In addition, with the chromatin immunocleavage sequencing assay, we observed a significant enrichment of RUNX1/CBFß-SMMHC target genes in Runx1f/fMx1-CreCbfb+/56M cells, especially among downregulated genes, suggesting that RUNX1 and CBFß-SMMHC mainly function together as activators of gene expression through direct target gene binding. These data indicate that Runx1 is indispensable for Cbfb-MYH11-induced leukemogenesis by working together with CBFß-SMMHC to regulate critical genes associated with the generation of a functional AMP population.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

IL-33/IL1RL1 axis regulates cell survival through the p38 MAPK pathway in acute myeloid leukemia.

Acute myeloid leukemia (AML) is often characterized by the presence of specific and recurrent chromosomal abnormalities. Current treatments have greatly increased remission rate, but relapse still occurs. Therefore, novel therapeutic approaches are required. Previously, using a conditional Cbfb-MYH11 knockin mouse model, we showed that Cbfb-MYH11 induces the expression of a cytokine receptor, IL1RL1. Treatment with IL-33, the only known ligand of IL1RL1, promotes leukemia cell survival in vitro. We further found that IL1RL1+ cells survive better with chemotherapy than IL1RL1- population. However, the mechanism is not clear. Here, we show that IL-33 treatment decreased drug sensitivity in the human inv(16) AML cell line ME-1. By RT-PCR, we found that IL-33 increased the expression of IL-4 and IL-6 and led to the activation of both p38 MAPK and NF-κB. We also showed that IL-33 decreased apoptosis with increased phosphorylation of p38 MAPK. Moreover, pre-treatment with MAPK inhibitor attenuated the phosphorylation of p38 enhanced by IL-33 and reversed the anti-apoptotic effect by IL-33. Taken together, our findings give news insights into the potential mechanism of the anti-apoptotic effect by IL-33/IL1RL1 axis in AML which will help in future drug development.

Use of polymeric CXCR4 inhibitors as siRNA delivery vehicles for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults and is associated with poor long-term survival often owing to relapse. Current treatments for AML are associated with considerable toxicity and are frequently not effective after relapse. Thus, it is important to develop novel therapeutic strategies. Short interfering RNA (siRNA)-based therapeutics targeting key oncogenes have been proposed as treatments for AML. We recently developed novel siRNA delivery polycations (PCX) based on AMD3100 (plerixafor), an FDA-approved inhibitor of the CXC chemokine receptor 4 (CXCR4). Inhibitors of CXCR4 have been shown to sensitize leukemia cells to chemotherapy. Therefore, PCX has the potential to target leukemia cells via two mechanisms: inhibition of CXCR4 and delivery of siRNAs against critical genes. In this report, we show that PCX exerts a cytotoxic effect on leukemia cells more effectively than other CXCR4 inhibitors, including AMD3100. In addition, we show that PCX can deliver siRNAs against the transcription factor RUNX1 to mouse and human leukemia cells. Overall, our study provides the first evidence that dual-function PCX/siRNA nanoparticles can simultaneously inhibit CXCR4 and deliver siRNAs, targeting key oncogenes in leukemia cells and that PCX/siRNA has clinical potential for the treatment of AML.

Loss of FBXO9 Enhances Proteasome Activity and Promotes Aggressiveness in Acute Myeloid Leukemia.

The hematopoietic system is maintained throughout life by stem cells that are capable of differentiating into all hematopoietic lineages. An intimate balance between self-renewal, differentiation, and quiescence is required to maintain hematopoiesis and disruption of this balance can result in malignant transformation. FBXO9, the substrate recognition component from the SCF E3 ubiquitin ligase family, is downregulated in patients with acute myeloid leukemia (AML) compared to healthy bone marrow, and this downregulation is particularly evident in patients with inv(16) AML. To study FBXO9 in malignant hematopoiesis, we generated a conditional knockout mouse model using a novel CRISPR/Cas9 strategy. Deletion of Fbxo9 in the murine hematopoietic system showed no adverse effects on stem and progenitor cell function but in AML lead to markedly accelerated and aggressive leukemia development in mice with inv(16). Not only did Fbxo9 play a role in leukemia initiation but it also functioned to maintain AML activity and promote disease progression. Quantitative mass spectrometry from primary tumors reveals tumors lacking Fbxo9 highly express proteins associated with metastasis and invasion as well as components of the ubiquitin proteasome system. We confirmed that the loss of FBXO9 leads to increased proteasome activity and tumors cells were more sensitive to in vitro proteasome inhibition with bortezomib, suggesting that FBXO9 expression may predict patients' response to bortezomib.

HDAC1 Is a Required Cofactor of CBFß-SMMHC and a Potential Therapeutic Target in Inversion 16 Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a neoplastic disease characterized by the uncontrolled proliferation and accumulation of immature myeloid cells. A common mutation in AML is the inversion of chromosome 16 [inv (16)], which generates a fusion between the genes for core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11), forming the oncogene CBFB-MYH11. The expressed protein, CBFß-SMMHC, forms a heterodimer with the key hematopoietic transcription factor RUNX1. Although CBFß-SMMHC was previously thought to dominantly repress RUNX1, recent work suggests that CBFß-SMMHC functions together with RUNX1 to activate transcription of specific target genes. However, the mechanism of this activity or a requirement for additional cofactors is not known. Here, we show that the epigenetic regulator histone deacetylase 1 (HDAC1) forms a complex with CBFß-SMMHC, colocalizes with RUNX1 and CBFß-SMMHC on the promoters of known fusion protein target genes, and that Hdac1 is required for expression of these genes. These results imply that HDAC1 is an important component of the CBFß-SMMHC transcriptional complex, and that leukemia cells expressing the fusion protein may be sensitive to treatment with HDAC1 inhibitors. Using a knock-in mouse model expressing CBFß-SMMHC, we found that in vivo treatment with the HDAC1 inhibitor entinostat decreased leukemic burden, and induced differentiation and apoptosis of leukemia cells. Together, these results demonstrate that HDAC1 is an important cofactor of CBFß-SMMHC and a potential therapeutic target in inv (16) AML. IMPLICATIONS: This report describes a novel role for HDAC1 as a cofactor for the leukemogenic fusion protein CBFß-SMMHC and shows that inhibitors of HDAC1 effectively target leukemia cells expressing the fusion protein in vivo.

IL1RL1 is dynamically expressed on Cbfb-MYH11+ leukemia stem cells and promotes cell survival.

Acute myeloid leukemia (AML) is often characterized by the presence of specific, recurrent chromosomal abnormalities. One of the most common aberrations, inversion of chromosome 16 [inv(16)], generates the fusion oncogene CBFB-MYH11. Previously, we used a mouse knock-in model to show that Cbfb-MYH11 induces changes in gene expression and results in the accumulation of abnormal myeloid cells, a subset of which are enriched for leukemia stem cell (LSC) activity. One gene upregulated by Cbfb-MYH11 encodes the cytokine receptor IL1RL1 (ST2). IL1RL1 and its ligand IL-33 are known regulators of mature myeloid cells, but their roles in AML are not known. Here, we use Cbfb-MYH11 knock-in mice to show that IL1RL1 is expressed by cell populations with high LSC activity, and that the cell surface expression of IL1RL1 is dynamic, implying that the expression of IL1RL1 is not restricted to a specific stage of differentiation. We also show that treatment with IL-33 increased serial replating ability and expression of pro-survival proteins in vitro. Finally, we show that IL1RL1+ cells can survive chemotherapy better than IL1RL1- cells in vivo. Collectively, our results indicate that IL1RL1 is dynamically expressed in Cbfb-MYH11+ leukemia cells and promotes their survival.

Chd7 deficiency delays leukemogenesis in mice induced by Cbfb-MYH11.

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. Previous studies showed that the interaction between CBFß-smooth muscle myosin heavy chain (SMMHC; encoded by CBFB-MYH11) and RUNX1 plays a critical role in the pathogenesis of this leukemia. Recently, it was shown that chromodomain helicase DNA-binding protein-7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 is also critical for CBFB-MYH11-induced leukemogenesis. To test this hypothesis, we generated Chd7f/fMx1-CreCbfb+/56M mice, which expressed the Cbfb-MYH11 fusion gene and deactivated Chd7 in hematopoietic cells upon inducing Cre with polyinosinic-polycytidylic acid. The Lin-Sca1-c-Kit+ (LK) population was significantly lower in Chd7f/fMx1-CreCbfb+/56M mice than in Mx1-CreCbfb+/56M mice. In addition, there were fewer 5-bromo-2'-deoxyuridine-positive cells in the LK population in Chd7f/fMx1-CreCbfb+/56M mice, and genes associated with cell cycle, cell growth, and proliferation were differentially expressed between Chd7f/fMx1-CreCbfb+/56M and Mx1-CreCbfb+/56M leukemic cells. In vitro studies showed that CHD7 interacted with CBFß-SMMHC through RUNX1 and that CHD7 enhanced transcriptional activity of RUNX1 and CBFß-SMMHC on Csf1r, a RUNX1 target gene. Moreover, RNA sequencing of c-Kit+ cells showed that CHD7 functions mostly through altering the expression of RUNX1 target genes. Most importantly, Chd7 deficiency delayed Cbfb-MYH11-induced leukemia in both primary and transplanted mice. These data indicate that Chd7 is important for Cbfb-MYH11-induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation.

RUNX1 and CBFß Mutations and Activities of Their Wild-Type Alleles in AML.

Mutations in RUNX1 and CBFB have long been recognized as important in hematological malignancies. Point mutations and deletions of RUNX1 are frequently found in myelodysplastic syndrome, myeloproliferative disease, and acute myeloid leukemia. Germline mutations in RUNX1 are associated with familial platelet disorder with predisposition to AML. In addition, as will be discussed in other chapters, both RUNX1 and CBFB are involved in recurrent chromosomal rearrangements in leukemia. More recently, roles for the non-mutated RUNX1 and CBFB genes have been identified in multiple leukemia subtypes. This chapter will discuss the roles of RUNX1 and CBFB, both in diseases caused by their mutations or deletions, as well as in the context of chromosomal rearrangements.

Targeting binding partners of the CBFß-SMMHC fusion protein for the treatment of inversion 16 acute myeloid leukemia.

Inversion of chromosome 16 (inv(16)) generates the CBFß-SMMHC fusion protein and is found in nearly all patients with acute myeloid leukemia subtype M4 with Eosinophilia (M4Eo). Expression of CBFß-SMMHC is causative for leukemia development, but the molecular mechanisms underlying its activity are unclear. Recently, there have been important advances in defining the role of CBFß-SMMHC and its binding partners, the transcription factor RUNX1 and the histone deacetylase HDAC8. Importantly, initial trials demonstrate that small molecules targeting these binding partners are effective against CBFß-SMMHC induced leukemia. This review will discuss recent advances in defining the mechanism of CBFß-SMMHC activity, as well as efforts to develop new therapies for inv(16) AML.

Germline PAX5 mutations and B cell leukemia.

The transcription factor PAX5 is required for normal B cell development and is frequently mutated or deleted in B cell precursor acute lymphoblastic leukemia (B-ALL). A new study demonstrates that germline hypomorphic mutations of PAX5 are associated with susceptibility to B-ALL, implicating PAX5 in a growing list of hematopoietic transcription factors mutated in familial leukemia predisposition syndromes.

The C-terminus of CBFß-SMMHC is required to induce embryonic hematopoietic defects and leukemogenesis.

The C-terminus of CBFß-SMMHC, the fusion protein produced by a chromosome 16 inversion in acute myeloid leukemia subtype M4Eo, contains domains for self-multimerization and transcriptional repression, both of which have been proposed to be important for leukemogenesis by CBFß-SMMHC. To test the role of the fusion protein's C-terminus in vivo, we generated knock-in mice expressing a C-terminally truncated CBFß-SMMHC (CBFß-SMMHCΔC95). Embryos with a single copy of CBFß-SMMHCΔC95 were viable and showed no defects in hematopoiesis, whereas embryos homozygous for the CBFß-SMMHCΔC95 allele had hematopoietic defects and died in mid-gestation, similar to embryos with a single-copy of the full-length CBFß-SMMHC. Importantly, unlike mice expressing full-length CBFß-SMMHC, none of the mice expressing CBFß-SMMHCΔC95 developed leukemia, even after treatment with a mutagen, although some of the older mice developed a nontransplantable myeloproliferative disease. Our data indicate that the CBFß-SMMHC's C-terminus is essential to induce embryonic hematopoietic defects and leukemogenesis.

Identification of benzodiazepine Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput screen against RUNX1-CBFß interaction.

Core binding factor (CBF) leukemias, those with translocations or inversions that affect transcription factor genes RUNX1 or CBFB, account for ~24% of adult acute myeloid leukemia (AML) and 25% of pediatric acute lymphocytic leukemia (ALL). Current treatments for CBF leukemias are associated with significant morbidity and mortality, with a 5-y survival rate of ~50%. We hypothesize that the interaction between RUNX1 and CBFß is critical for CBF leukemia and can be targeted for drug development. We developed high-throughput AlphaScreen and time-resolved fluorescence resonance energy transfer (TR-FRET) methods to quantify the RUNX1-CBFß interaction and screen a library collection of 243,398 compounds. Ro5-3335, a benzodiazepine identified from the screen, was able to interact with RUNX1 and CBFß directly, repress RUNX1/CBFB-dependent transactivation in reporter assays, and repress runx1-dependent hematopoiesis in zebrafish embryos. Ro5-3335 preferentially killed human CBF leukemia cell lines, rescued preleukemic phenotype in a RUNX1-ETO transgenic zebrafish, and reduced leukemia burden in a mouse CBFB-MYH11 leukemia model. Our data thus confirmed that RUNX1-CBFß interaction can be targeted for leukemia treatment and we have identified a promising lead compound for this purpose.

The role of microRNAs in acute myeloid leukemia.

MicroRNAs (miRs) are short (18-22 nucleotides) non-coding RNAs that are important in regulating gene expression. MiR expression is deregulated in many types of cancers, including leukemias. In acute myeloid leukemia (AML), the expression of specific miRs has been linked with both prognostically and cytogenetically defined subgroups. Recent studies have shown that deregulation of miR expression is not simply a consequence of AML but a potential contributer to leukemogenesis. This commentary will focus on select findings that describe the different mechanistic roles for miRs in the development of leukemia.

RUNX1 repression-independent mechanisms of leukemogenesis by fusion genes CBFB-MYH11 and AML1-ETO (RUNX1-RUNX1T1).

The core binding factor (CBF) acute myeloid leukemias (AMLs) are a prognostically distinct subgroup that includes patients with the inv(16) and t(8:21) chromosomal rearrangements. Both of these rearrangements result in the formation of fusion proteins, CBFB-MYH11 and AML1-ETO, respectively, that involve members of the CBF family of transcription factors. It has been proposed that both of these fusion proteins function primarily by dominantly repressing normal CBF transcription. However, recent reports have indicted that additional, CBF-repression independent activities may be equally important during leukemogenesis. This article will focus on these recent advances.