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.

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.

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.

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.