Jennifer L. Snead

A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib

In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph(+) metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34(+) cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph(+) metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated beta-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.

Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors

Acute dasatinib exposure commits Bcr-Abl-dependent cells to apoptosis

Pioneering work with the Bcr-Abl inhibitor, imatinib, demonstrated a requirement for constant Bcr-Abl inhibition to achieve maximal therapeutic benefit in treating chronic myeloid leukemia (CML), establishing a paradigm that has guided further drug development for this disease. Surprisingly, the second-generation Bcr-Abl inhibitor, dasatinib, was reported to be clinically effective with once-daily dosing, despite a short (3- to 5-hour) plasma half-life. Consistent with this observation, dasatinib treatment of progenitor cells from chronic-phase CML patients for 4 hours, followed by washout, or continuously for 72 hours both resulted in an induction of apoptosis and a reduction in the number of clonogenic cells. Such acute treatments with clinically achievable dasatinib concentrations also irreversibly committed Bcr-Abl+ CML cell lines to apoptotic cell death. Potent transient Bcr-Abl inhibition using the alternative inhibitor, nilotinib, also resulted in cell death. These findings demonstrate that in vitro assays designed to model in vivo pharmacokinetics can predict clinical efficacy. Furthermore, they challenge the widely held notion that continuous target inhibition is required for optimal efficacy of kinase inhibitors.

An image-based screen identifies a small molecule regulator of megakaryopoiesis

Molecules that control the lineage commitment of hematopoietic stem cells (HSCs) may allow the expansion of enriched progenitor populations for both research and therapeutic uses. In an effort to better understand and control the differentiation of HSCs to megakaryocytes, we carried out an image-based screen of a library of 50,000 heterocycles using primary human CD34+ cells. A class of naphthyridinone derivatives was identified that induces the differentiation of common myeloid progenitors (CMP) to megakaryocytes. Kinase profiling and subsequent functional assays revealed that these compounds act through inhibition of platelet-derived growth factor receptor (PDGFR) signaling in CMPs. Such molecules may ultimately have clinical utility in the treatment of thrombocytopenia.

New strategies for the first-line treatment of chronic myeloid leukemia: can resistance be avoided?

Imatinib is well established as a safe, effective therapy for patients with chronic myeloid leukemia (CML). However, point mutations in the kinase domain of Bcr-Abl can lead to imatinib resistance and reactivation of kinase activity. The second-generation Abl kinase inhibitors nilotinib and dasatinib were developed to reestablish disease control. A rising clinical challenge is using imatinib and novel Abl kinase inhibitors with the aim of completely preempting resistance. Fortunately, relapse on imatinib therapy so far has affected a minority of patients commencing treatment in the chronic phase of CML, and relapse rates continue to decline with treatment duration. In contrast, nearly all patients with CML have molecularly detectable disease. Thus, even among the best responders to imatinib, disease eradication is not achieved within a timeframe of years. Herein, we review current and emerging paradigms for using Abl kinase inhibitors to achieve maximal disease control and strategies to eradicate disease by targeting leukemic stem cells.

A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery:

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells

CRISPR–Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells—an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR–Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.Guide Swap challenges the hypothesis that Cas9–sgRNA binding is irreversible. The authors find that instead, nontargeting sgRNAs are swapped for targeting sgRNAs in the Cas9 complex. The method allows genome-scale functional screens in primary cells