Dual role of signaling pathways in myeloma requires cell-type specific targeting of ligand-receptor interactions.

Although most patients with multiple myeloma respond to treatment initially, therapy resistance develops almost invariably and only a subset of patients show durable responses to immunomodulatory (IMiD) therapies. While the immune microenvironment has been extensively studied in myeloma patients, its composition is currently not used as prognostic markers in clinical routine. We hypothesized that the outcome of immune signaling pathway engagement can be highly variable, depending on which two cellular populations participate in this interaction. This would have important prognostic and therapeutic implications, suggesting that it is crucial for immune pathways to be targeted in a specific cellular context. To test this hypothesis, we investigated a cohort of 27 patients with newly diagnosed multiple myeloma. We examined the complex regulatory networks within the immune compartment and their impact on disease progression. Analysis of immune cell composition and expression profiles revealed significant differences in the B cell compartment associated with treatment response. Transcriptional states in patients with short time to progression demonstrated an enrichment of pathways promoting B cell differentiation and inflammatory responses, which may indicate immune dysfunction. Importantly, the analysis of molecular interactions within the immune microenvironment highlights the dual role of signaling pathways, which can either be associated with good or poor prognosis depending on the cell types involved. Our findings therefore argue that therapeutic strategies targeting ligand-receptor interactions should take into consideration the composition of the microenvironment and the specific cell types involved in molecular interactions.

Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.

Identification of an Epi-metabolic dependency on EHMT2/G9a in T-cell acute lymphoblastic leukemia.

Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in acute lymphoblastic leukemia (ALL), suggesting new opportunities for therapeutic interventions. In this study, we identified G9a/EHMT2 as a potential target in T-ALL through the intersection of epigenome-centered shRNA and chemical screens. We subsequently validated G9a with low-throughput CRISPR-Cas9-based studies targeting the catalytic G9a SET-domain and the testing of G9a chemical inhibitors in vitro, 3D, and in vivo T-ALL models. Mechanistically we determined that G9a repression promotes lysosomal biogenesis and autophagic degradation associated with the suppression of sestrin2 (SESN2) and inhibition of glycogen synthase kinase-3 (GSK-3), suggesting that in T-ALL glycolytic dependent pathways are at least in part under epigenetic control. Thus, targeting G9a represents a strategy to exhaust the metabolic requirement of T-ALL cells.

Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19+ B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T cell-leukemia cell interface followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing, we demonstrated that a subset of refractory CD19low cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the Bruton's tyrosine kinase inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity. See related Spotlight by Zhao and Melenhorst, p. 1040.

Cell-free DNA for the detection of emerging treatment failure in relapsed/ refractory multiple myeloma.

Interrogation of cell-free DNA (cfDNA) represents an emerging approach to non-invasively estimate disease burden in multiple myeloma (MM). Here, we examined low-pass whole genome sequencing (LPWGS) of cfDNA for its predictive value in relapsed/ refractory MM (RRMM). We observed that cfDNA positivity, defined as ≥10% tumor fraction by LPWGS, was associated with significantly shorter progression-free survival (PFS) in an exploratory test cohort of 16 patients who were actively treated on diverse regimens. We prospectively determined the predictive value of cfDNA in 86 samples from 45 RRMM patients treated with elotuzumab, pomalidomide, bortezomib, and dexamethasone in a phase II clinical trial (NCT02718833). PFS in patients with tumor-positive and -negative cfDNA after two cycles of treatment was 1.6 and 17.6 months, respectively (HR 7.6, P < 0.0001). Multivariate hazard modelling confirmed cfDNA as independent risk factor (HR 96.6, P = 6.92e-05). While correlating with serum-free light chains and bone marrow, cfDNA additionally discriminated patients with poor PFS among those with the same response by IMWG criteria. In summary, detectability of MM-derived cfDNA, as a measure of substantial tumor burden with therapy, independently predicts poor PFS and may provide refinement for standard-of-care response parameters to identify patients with poor response to treatment earlier than is currently feasible.

ETV6-NCOA2 fusion induces T/myeloid mixed-phenotype leukemia through transformation of nonthymic hematopoietic progenitor cells.

Mixed-phenotype acute leukemia is a rare subtype of leukemia in which both myeloid and lymphoid markers are co-expressed on the same malignant cells. The pathogenesis is largely unknown, and the treatment is challenging. We previously reported the specific association of the recurrent t(8;12)(q13;p13) chromosomal translocation that creates the ETV6-NCOA2 fusion with T/myeloid leukemias. Here we report that ETV6-NCOA2 initiates T/myeloid leukemia in preclinical models; ectopic expression of ETV6-NCOA2 in mouse bone marrow hematopoietic progenitors induced T/myeloid lymphoma accompanied by spontaneous Notch1-activating mutations. Similarly, cotransduction of human cord blood CD34+ progenitors with ETV6-NCOA2 and a nontransforming NOTCH1 mutant induced T/myeloid leukemia in immunodeficient mice; the immunophenotype and gene expression pattern were similar to those of patient-derived ETV6-NCOA2 leukemias. Mechanistically, we show that ETV6-NCOA2 forms a transcriptional complex with ETV6 and the histone acetyltransferase p300, leading to derepression of ETV6 target genes. The expression of ETV6-NCOA2 in human and mouse nonthymic hematopoietic progenitor cells induces transcriptional dysregulation, which activates a lymphoid program while failing to repress the expression of myeloid genes such as CSF1 and MEF2C. The ETV6-NCOA2 induced arrest at an early immature T-cell developmental stage. The additional acquisition of activating NOTCH1 mutations transforms the early immature ETV6-NCOA2 cells into T/myeloid leukemias. Here, we describe the first preclinical model to depict the initiation of T/myeloid leukemia by a specific somatic genetic aberration.

Dynamic transcriptional reprogramming leads to immunotherapeutic vulnerabilities in myeloma.

While there is extensive evidence for genetic variation as a basis for treatment resistance, other sources of variation result from cellular plasticity. Using multiple myeloma as an example of an incurable lymphoid malignancy, we show how cancer cells modulate lineage restriction, adapt their enhancer usage and employ cell-intrinsic diversity for survival and treatment escape. By using single-cell transcriptome and chromatin accessibility profiling, we show that distinct transcriptional states co-exist in individual cancer cells and that differential transcriptional regulon usage and enhancer rewiring underlie these alternative transcriptional states. We demonstrate that exposure to standard treatment further promotes transcriptional reprogramming and differential enhancer recruitment while simultaneously reducing developmental potential. Importantly, treatment generates a distinct complement of actionable immunotherapy targets, such as CXCR4, which can be exploited to overcome treatment resistance. Our studies therefore delineate how to transform the cellular plasticity that underlies drug resistance into immuno-oncologic therapeutic opportunities.

Single-Cell Profiling Reveals Metabolic Reprogramming as a Resistance Mechanism in BRAF-Mutated Multiple Myeloma.

PURPOSE: Although remarkably effective in some patients, precision medicine typically induces only transient responses despite initial absence of resistance-conferring mutations. Using BRAF-mutated myeloma as a model for resistance to precision medicine we investigated if BRAF-mutated cancer cells have the ability to ensure their survival by rapidly adapting to BRAF inhibitor treatment. EXPERIMENTAL DESIGN: Full-length single-cell RNA (scRNA) sequencing (scRNA-seq) was conducted on 3 patients with BRAF-mutated myeloma and 1 healthy donor. We sequenced 1,495 cells before, after 1 week, and at clinical relapse to BRAF/MEK inhibitor treatment. We developed an in vitro model of dabrafenib resistance using genetically homogeneous single-cell clones from two cell lines with established BRAF mutations (U266, DP6). Transcriptional and epigenetic adaptation in resistant cells were defined by RNA-seq and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). Mitochondrial metabolism was characterized by metabolic flux analysis. RESULTS: Profiling by scRNA-seq revealed rapid cellular state changes in response to BRAF/MEK inhibition in patients with myeloma and cell lines. Transcriptional adaptation preceded detectable outgrowth of genetically discernible drug-resistant clones and was associated with widespread enhancer remodeling. As a dominant vulnerability, dependency on oxidative phosphorylation (OxPhos) was induced. In treated individuals, OxPhos was activated at the time of relapse and showed inverse correlation to MAPK activation. Metabolic flux analysis confirmed OxPhos as a preferential energetic resource of drug-persistent myeloma cells. CONCLUSIONS: This study demonstrates that cancer cells have the ability to rapidly adapt to precision treatments through transcriptional state changes, epigenetic adaptation, and metabolic rewiring, thus facilitating the development of refractory disease while simultaneously exposing novel vulnerabilities.

Single-cell RNA-seq reveals developmental plasticity with coexisting oncogenic states and immune evasion programs in ETP-ALL.

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.

Tracking myeloma tumor DNA in peripheral blood.

Over the past years, the emergence of liquid biopsy technologies has dramatically expanded our ability to assess multiple myeloma without the need for invasive sampling. Interrogation of cell-free DNA from the peripheral blood recapitulates the mutational landscape at excellent concordance with matching bone marrow aspirates. It can quantify disease burden and identify previously undetected resistance mechanisms which may inform clinical management in real-time. The convenience of sample acquisition and storage provides strong procedural benefits over currently available testing. Further investigations will have to define the role of cell-free DNA as a diagnostic measure by determining clinically relevant tumor thresholds in comparison to existing routine parameters. This review presents an overview of currently available assays and discusses the clinical value, potential and limitations of cell-free DNA technologies for the assessment of this challenging disease.

PRC2 loss induces chemoresistance by repressing apoptosis in T cell acute lymphoblastic leukemia.

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming. Here, we report that mitochondrial apoptosis resistance in T cell acute lymphoblastic leukemia (T-ALL) is mediated by inactivation of polycomb repressive complex 2 (PRC2). In T-ALL clinical specimens, loss-of-function mutations of PRC2 core components (EZH2, EED, or SUZ12) were associated with mitochondrial apoptosis resistance. In T-ALL cells, PRC2 depletion induced resistance to apoptosis induction by multiple chemotherapeutics with distinct mechanisms of action. PRC2 loss induced apoptosis resistance via transcriptional up-regulation of the LIM domain transcription factor CRIP2 and downstream up-regulation of the mitochondrial chaperone TRAP1 These findings demonstrate the importance of mitochondrial apoptotic priming as a prognostic factor in T-ALL and implicate mitochondrial chaperone function as a molecular determinant of chemotherapy response.

Zebrafish blastomere screen identifies retinoic acid suppression of MYB in adenoid cystic carcinoma.

Pluripotent cells have been used to probe developmental pathways that are involved in genetic diseases and oncogenic events. To find new therapies that would target MYB-driven tumors, we developed a pluripotent zebrafish blastomere culture system. We performed a chemical genetic screen and identified retinoic acid agonists as suppressors of c-myb expression. Retinoic acid treatment also decreased c-myb gene expression in human leukemia cells. Translocations that drive overexpression of the oncogenic transcription factor MYB are molecular hallmarks of adenoid cystic carcinoma (ACC), a malignant salivary gland tumor with no effective therapy. Retinoic acid agonists inhibited tumor growth in vivo in ACC patient-derived xenograft models and decreased MYB binding at translocated enhancers, thereby potentially diminishing the MYB positive feedback loop driving ACC. Our findings establish the zebrafish pluripotent cell culture system as a method to identify modulators of tumor formation, particularly establishing retinoic acid as a potential new effective therapy for ACC.

Comprehensive characterization of circulating and bone marrow-derived multiple myeloma cells at minimal residual disease.

The presence or absence of minimal residual disease (MRD) in patients with multiple myeloma (MM) has emerged as a useful marker to determine the depth of remission. MRD negativity as an endpoint has been shown to be associated with improved progression-free survival in many studies. MRD detection is therefore part of numerous clinical trial protocols for MM. At the present time, two methodologies are most widely accepted for MRD detection: (1) multicolor flow cytometry and (2) next-generation sequencing-based clonotype detection. While both of those methodologies enable accurate quantification of MRD in the bone marrow (BM), with sensitivity as low as 10-5 to 10-6, there are several limitations to these methods. First, these approaches reveal the presence or absence of MRD but provide limited molecular information about MM. More comprehensive characterization of MM cells at the MRD stage may identify molecular mechanisms of drug resistance. Second, MRD detection in the BM is typically performed at one time point only, but more frequent detection may define the duration of the MRD status and thus refine its prognostic value. Third, less-invasive approaches that avoid the discomfort and risk associated with BM biopsy would be highly desirable, especially in elderly or frail patients. "Liquid biopsy" for the detection and characterization of circulating MM cells may address these issues. Although MRD detection in the peripheral blood at the same sensitivity as in the BM may be challenging, the identification of patients who do not achieve MRD negativity might reduce the need for BM biopsies. Here, we give an overview of approaches that have been described to detect and characterize MM cells when they occur at very low frequencies in the peripheral blood or in the BM, emphasizing recently described next-generation sequencing approaches for more comprehensive characterization of circulating MM cells.

Peptidomimetic blockade of MYB in acute myeloid leukemia.

Aberrant gene expression is a hallmark of acute leukemias. MYB-driven transcriptional coactivation with CREB-binding protein (CBP)/P300 is required for acute lymphoblastic and myeloid leukemias, including refractory MLL-rearranged leukemias. Using structure-guided molecular design, we developed a peptidomimetic inhibitor MYBMIM that interferes with the assembly of the molecular MYB:CBP/P300 complex and rapidly accumulates in the nuclei of AML cells. Treatment of AML cells with MYBMIM led to the dissociation of the MYB:CBP/P300 complex in cells, its displacement from oncogenic enhancers enriched for MYB binding sites, and downregulation of MYB-dependent gene expression, including of MYC and BCL2 oncogenes. AML cells underwent mitochondrial apoptosis in response to MYBMIM, which was partially rescued by ectopic expression of BCL2. MYBMIM impeded leukemia growth and extended survival of immunodeficient mice engrafted with primary patient-derived MLL-rearranged leukemia cells. These findings elucidate the dependence of human AML on aberrant transcriptional coactivation, and establish a pharmacologic approach for its therapeutic blockade.

Genetic interrogation of circulating multiple myeloma cells at single-cell resolution.

Multiple myeloma (MM) remains an incurable disease, with a treatment-refractory state eventually developing in all patients. Constant clonal evolution and genetic heterogeneity of MM are a likely explanation for the emergence of drug-resistant disease. Monitoring of MM genomic evolution on therapy by serial bone marrow biopsy is unfortunately impractical because it involves an invasive and painful procedure. We describe how noninvasive and highly sensitive isolation and characterization of circulating tumor cells (CTCs) from peripheral blood at single-cell resolution recapitulate MM in the bone marrow. We demonstrate that CTCs provide the same genetic information as bone marrow MM cells and even reveal mutations with greater sensitivity than bone marrow biopsies in some cases. Single CTC RNA sequencing enables classification of MM and quantitative assessment of genes that are relevant for prognosis. We propose that the genomic characterization of CTCs should be included in clinical trials to follow the emergence of resistant subclones after MM therapy.

An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma.

Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.

Metabolic Mechanisms of Drug Resistance in Leukemia.

Tumor cells frequently undergo metabolic reprogramming, but it is unknown how these metabolic changes relate to drug resistance. A recent article now demonstrates that PI3K/AKT signaling causes a metabolic switch from glutaminolysis to aerobic glycolysis in Notch-dependent T cell acute lymphoblastic leukemia (T-ALL).

Complete hematologic response of early T-cell progenitor acute lymphoblastic leukemia to the γ-secretase inhibitor BMS-906024: genetic and epigenetic findings in an outlier case.

Notch pathway antagonists such as γ-secretase inhibitors (GSIs) are being tested in diverse cancers, but exceptional responses have yet to be reported. We describe the case of a patient with relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) who achieved a complete hematologic response following treatment with the GSI BMS-906024. Whole-exome sequencing of leukemic blasts revealed heterozygous gain-of-function driver mutations in NOTCH1, CSF3R, and PTPN11, and a homozygous/hemizygous loss-of-function mutation in DNMT3A. The three gain-of-function mutations were absent from remission marrow cells, but the DNMT3A mutation persisted in heterozygous form in remission marrow, consistent with an origin for the patient's ETP-ALL from clonal hematopoiesis. Ex vivo culture of ETP-ALL blasts confirmed high levels of activated NOTCH1 that were repressed by GSI treatment, and RNA-seq documented that GSIs downregulated multiple known Notch target genes. Surprisingly, one potential target gene that was unaffected by GSIs was MYC, a key Notch target in GSI-sensitive T-ALL of cortical T-cell type. H3K27ac super-enhancer landscapes near MYC showed a pattern previously reported in acute myeloid leukemia (AML) that is sensitive to BRD4 inhibitors, and in line with this ETP-ALL blasts downregulated MYC in response to the BRD4 inhibitor JQ1. To our knowledge, this is the first example of complete response of a Notch-mutated ETP-ALL to a Notch antagonist and is also the first description of chromatin landscapes associated with ETP-ALL. Our experience suggests that additional attempts to target Notch in Notch-mutated ETP-ALL are merited.

EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma.

The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation.