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.

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.

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.

A single dividing cell population with imbalanced fate drives oesophageal tumour growth.

Understanding the cellular mechanisms of tumour growth is key for designing rational anticancer treatment. Here we used genetic lineage tracing to quantify cell behaviour during neoplastic transformation in a model of oesophageal carcinogenesis. We found that cell behaviour was convergent across premalignant tumours, which contained a single proliferating cell population. The rate of cell division was not significantly different in the lesions and the surrounding epithelium. However, dividing tumour cells had a uniform, small bias in cell fate so that, on average, slightly more dividing than non-dividing daughter cells were generated at each round of cell division. In invasive cancers induced by Kras(G12D) expression, dividing cell fate became more strongly biased towards producing dividing over non-dividing cells in a subset of clones. These observations argue that agents that restore the balance of cell fate may prove effective in checking tumour growth, whereas those targeting cycling cells may show little selectivity.

Esophageal stem cells and genetics/epigenetics in esophageal cancer.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the relationship between stem cells, cancer, and the esophagus; the behavior of esophageal stem cells; and the role of genetics and epigenetics in approaches to translational research.

Mutation, clonal fitness and field change in epithelial carcinogenesis.

Developments in lineage tracing in mouse models have revealed how stem cells maintain normal squamous and glandular epithelia. Here we review recent quantitative studies tracing the fate of individual mutant stem cells which have uncovered how common oncogenic mutations alter cell behaviour, creating clones with a growth advantage that may persist long term. In the intestine this occurs by a mutant clone colonizing an entire crypt, whilst in the squamous oesophagus blocking differentiation creates clones that expand to colonize large areas of epithelium, a phenomenon known as field change. We consider the implications of these findings for early cancer evolution and the cancer stem cell hypothesis, and the prospects of targeted cancer prevention by purging mutant clones from normal-appearing epithelia.

Differentiation imbalance in single oesophageal progenitor cells causes clonal immortalization and field change.

Multiple cancers may arise from within a clonal region of preneoplastic epithelium, a phenomenon termed 'field change'. However, it is not known how field change develops. Here we investigate this question using lineage tracing to track the behaviour of scattered single oesophageal epithelial progenitor cells expressing a mutation that inhibits the Notch signalling pathway. Notch is frequently subject to inactivating mutation in squamous cancers. Quantitative analysis reveals that cell divisions that produce two differentiated daughters are absent from mutant progenitors. As a result, mutant clones are no longer lost by differentiation and become functionally immortal. Furthermore, mutant cells promote the differentiation of neighbouring wild-type cells, which are then lost from the tissue. These effects lead to clonal expansion, with mutant cells eventually replacing the entire epithelium. Notch inhibition in progenitors carrying p53 stabilizing mutations creates large confluent regions of doubly mutant epithelium. Field change is thus a consequence of imbalanced differentiation in individual progenitor cells.

Ovarian cancer: Ion channel and aquaporin expression as novel targets of clinical potential.

Ovarian cancer is associated with limited overall survival, due to problems in early detection and therapy. Membrane ion channels have been proposed to play a significant, concerted role in the cancer process, from initial proliferation to metastasis, and promise to be early, functional biomarkers. We review the evidence for ion channel and aquaporin expression and functioning in human ovarian cancer cells and tissues. In vitro, K(+) channels, mainly voltage-gated, including Ca(2+)-activated channels, have been found to control the cell cycle, as in other cancers. Voltage-gated, volume-regulated and intracellular Cl(-) channels have been detected in vitro and in vivo and shown to be involved in proliferation, adhesion and invasion. Evidence for 'transient receptor potential', voltage-gated sodium and calcium channels, which have been shown to contribute to pathogenesis of other carcinomas, is also emerging in ovarian cancer. Aquaporins may be involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. It is concluded that functional expression of ion channels and their regulation by steroid hormones and growth factors are an integral part of ovarian cancer development and progression. Furthermore, ion channels may be involved in multidrug resistance, commonly associated with treatment of ovarian cancer. We propose that ion channel studies can facilitate our understanding of the pathobiology of ovarian cancer and, ultimately, can serve as viable novel targets for its clinical management.