Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.

PP2A is a therapeutically targetable driver of cell fate decisions via a c-Myc/p21 axis in human and murine acute myeloid leukemia.

Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

The ROR1 antibody-drug conjugate huXBR1-402-G5-PNU effectively targets ROR1+ leukemia.

Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody-based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

Genomic analysis of cellular hierarchy in acute myeloid leukemia using ultrasensitive LC-FACSeq.

Hematopoiesis is hierarchical, and it has been postulated that acute myeloid leukemia (AML) is organized similarly with leukemia stem cells (LSCs) residing at the apex. Limited cells acquired by fluorescence activated cell sorting in tandem with targeted amplicon-based sequencing (LC-FACSeq) enables identification of mutations in small subpopulations of cells, such as LSCs. Leveraging this, we studied clonal compositions of immunophenotypically-defined compartments in AML through genomic and functional analyses at diagnosis, remission and relapse in 88 AML patients. Mutations involving DNA methylation pathways, transcription factors and spliceosomal machinery did not differ across compartments, while signaling pathway mutations were less frequent in putative LSCs. We also provide insights into TP53-mutated AML by demonstrating stepwise acquisition of mutations beginning from the preleukemic hematopoietic stem cell stage. In 10 analyzed cases, acquisition of additional mutations and del(17p) led to genetic and functional heterogeneity within the LSC pool with subclones harboring varying degrees of clonogenic potential. Finally, we use LC-FACSeq to track clonal evolution in serial samples, which can also be a powerful tool to direct targeted therapy against measurable residual disease. Therefore, studying clinically significant small subpopulations of cells can improve our understanding of AML biology and offers advantages over bulk sequencing to monitor the evolution of disease.

Targeted degradation of the enhancer lysine acetyltransferases CBP and p300.

The enhancer factors CREB-binding protein (CBP) and p300 (also known as KAT3A and KAT3B) maintain gene expression programs through lysine acetylation of chromatin and transcriptional regulators and by scaffolding functions mediated by several protein-protein interaction domains. Small molecule inhibitors that target some of these domains have been developed; however, they cannot completely ablate p300/CBP function in cells. Here we describe a chemical degrader of p300/CBP, dCBP-1. Leveraging structures of ligand-bound p300/CBP domains, we use in silico modeling of ternary complex formation with the E3 ubiquitin ligase cereblon to enable degrader design. dCBP-1 is exceptionally potent at killing multiple myeloma cells and can abolish the enhancer that drives MYC oncogene expression. As an efficient degrader of this unique class of acetyltransferases, dCBP-1 is a useful tool alongside domain inhibitors for dissecting the mechanism by which these factors coordinate enhancer activity in normal and diseased cells.

LC-FACSeq is a method for detecting rare clones in leukemia.

Detecting, characterizing, and monitoring rare populations of cells can increase testing sensitivity, give insight into disease mechanism, and inform clinical decision making. One area that can benefit from increased resolution is management of cancers in clinical remission but with measurable residual disease (MRD) by multicolor FACS. Detecting and monitoring genomic clonal resistance to treatment in the setting of MRD is technically difficult and resource intensive due to the limited amounts of disease cells. Here, we describe limited-cell FACS sequencing (LC-FACSeq), a reproducible, highly sensitive method of characterizing clonal evolution in rare cells relevant to different types of acute and chronic leukemias. We demonstrate the utility of LC-FACSeq for broad multigene gene panels and its application for monitoring sequential acquisition of mutations conferring therapy resistance and clonal evolution in long-term ibrutinib treatment of patients with chronic lymphocytic leukemia. This technique is generalizable for monitoring of other blood and marrow infiltrating cancers.

CLEAR: coverage-based limiting-cell experiment analysis for RNA-seq.

BACKGROUND: Direct cDNA preamplification protocols developed for single-cell RNA-seq have enabled transcriptome profiling of precious clinical samples and rare cell populations without the need for sample pooling or RNA extraction. We term the use of single-cell chemistries for sequencing low numbers of cells limiting-cell RNA-seq (lcRNA-seq). Currently, there is no customized algorithm to select robust/low-noise transcripts from lcRNA-seq data for between-group comparisons. METHODS: Herein, we present CLEAR, a workflow that identifies reliably quantifiable transcripts in lcRNA-seq data for differentially expressed genes (DEG) analysis. Total RNA obtained from primary chronic lymphocytic leukemia (CLL) CD5+ and CD5- cells were used to develop the CLEAR algorithm. Once established, the performance of CLEAR was evaluated with FACS-sorted cells enriched from mouse Dentate Gyrus (DG). RESULTS: When using CLEAR transcripts vs. using all transcripts in CLL samples, downstream analyses revealed a higher proportion of shared transcripts across three input amounts and improved principal component analysis (PCA) separation of the two cell types. In mouse DG samples, CLEAR identifies noisy transcripts and their removal improves PCA separation of the anticipated cell populations. In addition, CLEAR was applied to two publicly-available datasets to demonstrate its utility in lcRNA-seq data from other institutions. If imputation is applied to limit the effect of missing data points, CLEAR can also be used in large clinical trials and in single cell studies. CONCLUSIONS: lcRNA-seq coupled with CLEAR is widely used in our institution for profiling immune cells (circulating or tissue-infiltrating) for its transcript preservation characteristics. CLEAR fills an important niche in pre-processing lcRNA-seq data to facilitate transcriptome profiling and DEG analysis. We demonstrate the utility of CLEAR in analyzing rare cell populations in clinical samples and in murine neural DG region without sample pooling.

Mobile apps for professional dermatology education: an objective review.

Mobile dermatology applications (apps) created for the purpose of educating students and trainees present convenient supplemental learning opportunities. Before these apps can be widely utilized, there must be a method to assess educational objectives, quality, comprehensiveness of content, evidence-based accuracy, user-friendly design, and potential for bias. Herein, an established rubric was used to conduct a graded review of apps spanning general dermatology, skin cancer, and cosmetics, with an additional emphasis on affordability and accessibility for the user.

PI3K p110δ inactivation antagonizes chronic lymphocytic leukemia and reverses T cell immune suppression.

Targeted therapy with small molecules directed at essential survival pathways in leukemia represents a major advance, including the phosphatidylinositol-3'-kinase (PI3K) p110δ inhibitor idelalisib. Here, we found that genetic inactivation of p110δ (p110δD910A/D910A) in the Eµ-TCL1 murine chronic lymphocytic leukemia (CLL) model impaired B cell receptor signaling and B cell migration, and significantly delayed leukemia pathogenesis. Regardless of TCL1 expression, p110δ inactivation led to rectal prolapse in mice resembling autoimmune colitis in patients receiving idelalisib. Moreover, we showed that p110δ inactivation in the microenvironment protected against CLL and acute myeloid leukemia. After receiving higher numbers of TCL1 leukemia cells, half of p110δD910A/D910A mice spontaneously recovered from high disease burden and resisted leukemia rechallenge. Despite disease resistance, p110δD910A/D910A mice exhibited compromised CD4+ and CD8+ T cell response, and depletion of CD4+ or CD8+ T cells restored leukemia. Interestingly, p110δD910A/D910A mice showed significantly impaired Treg expansion that associated with disease clearance. Reconstitution of p110δD910A/D910A mice with p110δWT/WT Tregs reversed leukemia resistance. Our findings suggest that p110δ inhibitors may have direct antileukemic and indirect immune-activating effects, further supporting that p110δ blockade may have a broader immune-modulatory role in types of leukemia that are not sensitive to p110δ inhibition.

The Role of an Integrated Multidisciplinary Clinic in the Management of Patients with Cutaneous Lymphoma.

The clinical benefit of a multidisciplinary clinic practice model has been well described in a variety of medical specialties and cancer types. It proves particularly valuable when an integrated team is needed to optimally manage patients with rare or complex neoplasms. However, the ideal implementation of an integrated multidisciplinary care program for translational research and education has not been well reported. Herein, we propose how a multimodality cutaneous lymphoma (CL) clinic model can optimally manage CL patients. We offer our perspective on this model as an efficient means for delivering patient care, a continuing education resource for referring physicians, a conduit for translational and clinical research, and an educational tool for medical students, house staff, and fellows.