No abstract

The ability to assess antileukemic drug activity on primary patient samples is a powerful tool in determining potential drug targets and selection of therapeutic agents with biological and functional rationale. We previously established small molecule inhibitor screens for use on freshly isolated leukemia cells for this purpose. Here we describe a method that produces functional small molecule inhibitor screening results using cryopreserved primary acute myeloid leukemia cells. This method was established to take advantage of biorepositories containing archival material, such as those established by the Children's Oncology Group, and to enable validation of potential pathway dependencies uncovered by genomic analysis. Various conditions used to thaw and culture cryopreserved specimens were assessed for effect on viability, differentiation, and the ability to recapitulate sensitivity results obtained on fresh samples. The most reproducible results were obtained by quick-thawing and culturing samples in cytokine rich media before performing drug screens. Our data suggest that cytokine-enriched media aids in maintaining the viability and numbers required to perform functional analysis on cryopreserved leukemia cells. This method can aid in producing informative data on therapeutic targeting and precision medicine efforts in leukemia by making use of biorepositories and bio banks.

Acute Lymphoblastic Leukemia (ALL) harboring the t(17;19)(q22;p13) is a rare subtype of leukemia with a dismal prognosis. This recurring translocation produces an aberrant TCF3-HLF fusion with distinct gene expression profiles and drug sensitivity. Recent studies have shown that this subtype of ALL might be targeted using therapies inhibiting BCL-2 and the pre-B cell receptor through inhibition of SRC family kinases. However, preliminary validation of these studies have revealed significant heterogeneity of response to BCL-2 and SRC inhibitors. As such, we sought to identify other possible targets that could overcome this heterogeneity and improve response to therapy. Methods: One local as well as four other samples from the Children's Oncology Group's ALL Biorepository with TCF3-HLF ALL were expanded in immunodeficient NSG mice. All samples were verified by RT-PCR and Sanger sequencing for the fusion transcript. Samples were then interrogated with our functional drug screen that is comprised of compounds with activity against two-thirds of the tyrosine kinome as well as other non-tyrosine kinase pathways, including RAF/MEK/MAPKs, PI3K/AKT/mTOR, AMPK, ATM, Aurora kinases, CAMKs, CDKs, GSK3a/b, IKK, PKA, PKC, PLK1, and RAF as well as BCL2 family, BRD4, IDH1/2, Hedgehog, HSP90, NOTCH/g-secretase, proteasome, survivin, STAT3, and WNT/b-catenin. The samples were sequenced using the Agilent SureSelect Strand-Specific RNA Library Preparation Kit on the Bravo robot (Agilent). All five patient samples successfully engrafted into NSG mice and were tested for in vivo sensitivity as assessed for disease burden or survival. Results: Three patient samples were identified to carry Type I translocations fusing exon 13 of TCF3 with variable intronic insertions followed by exon 4 of HLF. All three type I translocations produced different fusions due to different lengths within the variable region. One sample predicted a truncation product of TCF3 ending in exon 13 with an early stop codon within the variable region. Two patient samples carried the identical type II translocation fusing Exon 12 of TCF3 with exon 4 of HLF. RNA-seq results of the five samples identified other individual translocations, but none involved other specific disease related lesions. Results from our drug screen showed significant heterogeneity in response to the majority of drugs assayed including the ABL/SRC inhibitor dasatinib and the BCL-2 inhibitor venetoclax. Further, in vivo studies exposing cohorts of animals to vehicle (n=5), dasatinib (40mg/kg/day; n=5), venetoclax (25-100mg/kg/day; n=5) or combination of dasatinib and venetoclax (n=5) identified only two samples with treatment benefit. Interestingly, review of the results of the drug screen suggested hypersensitivity to aurora kinase inhibitors. Each sample was tested in vivo in cohorts of vehicle (n=5) and alisertib (30mg/kg/day; n=5). All five ALL samples showed significant response (p<0.01 for all five samples compared to their respective vehicle controls by Chi Square analysis). All animals tolerated treatment and no animal showed significant hematologic toxicity from treatment with drugs. Conclusion: Our results suggest that TCF3-HLF ALL is a heterogeneous subset of ALL with both different gene expression patterns from TCF3-HLF to other fusions as well as functional drug response. In vivo validation in the murine model with these five samples suggests significant heterogeneity to current pursued targets such as BCL-2 and SRC compared to previously published reports. Most intriguing, all samples tested with alisertib identified significant in vivo response suggesting unique preclinical support to pursue further clinical testing within this rare and lethal subtype of ALL. Disclosures Leonard: Amgen: Research Funding. Mullighan:Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy. Tyner:Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding. Druker:GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Patient True Talk: Consultancy; Fred Hutchinson Cancer Research Center: Research Funding; ARIAD: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Celgene: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties.

Not available.

The ability to assess anti-leukemic activity of drugs on patient samples is a powerful tool in determining potential drug targets and may enhance selection of therapeutic agents with a biologic and functional rationale for individual patients. We have previously optimized small-molecule screens performed on freshly isolated leukemia cells for this purpose. Here we describe a novel method we have recently developed that produces reliable functional testing results from previously frozen specimens. This method was established to take advantage of bio-repositories containing archival primary leukemia specimens and to aid in the validation of potential drug target analysis and pathway activation. Three different culture media comprising various cocktails of cytokines were tested for their ability to maintain cell viability after thawing and to produce inhibitor results similar to those obtained using freshly isolated acute myeloid leukemia (AML) cells. We identified a method of thawing cells by first culturing for 24-48 hours in cytokine-enriched medium (RPMI-1640, 10% fetal bovine serum (FBS), and low concentrations of GM-CSF, G-CSF, SCF, and IL-3) prior to plating in RPMI-1640 and 10% FBS (R10). This method maintained high cell viability and produced inhibitor results comparable to those of freshly isolated cells plated in R10. Half-maximal inhibitory concentration (IC50) analysis was used to determine the relative success of the thaw process and reliability of results. Comparison of data produced from frozen cells initially cultured in cytokine-enriched medium to data obtained from freshly isolated cells showed few differences in effective drug hits. To examine the effects of freeze-thawing and cytokines on cell subpopulations, we performed FACS analysis on 8 markers to examine the level of cell differentiation under each condition. FACS sorting revealed increases in expression of myeloid differentiation markers (CD11b and CD14) after thaw in all media tested compared to fresh cells in cultured in R10. Thawed cells grown in cytokine-enriched medium remained closest to fresh controls as measured by antigen expression, including CD11b and CD14. Cell viability post-thaw was a strong indicator of samples that would fail to produce reliable inhibitor results. We observed that post-thaw viability <80% served as a strong indicator of unreliable inhibitor data, even if dead cells were removed prior to plating. Cells cultured in cytokine-enriched medium for 24-48 hours resulted in 11-33% higher viability prior to plating in R10. Percent median IC50 for specific inhibitors assayed on fresh and frozen samples showed similar trends in effective drugs (Fig. 1A). Discrepancies observed in fresh and frozen sample data may be due to inconsistent fresh sample data as we demonstrated that frozen samples were highly consistent compared to other frozen samples (Fig. 1B). Lower R2 values could be attributed to variability observed with non-effective drugs. Taken together, our data indicate that thawing leukemia specimens in cytokine-enriched media can allow for the generation of informative small-molecule inhibitor screening data and provide a novel method for making use of archived primary human leukemia specimens for further downstream functional analyses. Figure 1. Median IC50 Comparison. Median IC50 values are calculated from >3000 samples IC50 values. Percent median is the percent a particular sample IC50 compares to the median of all samples of that disease character. Mean values are shown for inhibitors with replicates. R2 from raw IC50. (A) Comparison of effective drug hits on fresh and frozen primary peripheral blood AML samples (R2 = 0.8151). (B) Comparison of effective drug hits obtained on a freshly isolated AML bone marrow sample versus 2 matched frozen/thawed samples from peripheral blood. Fresh vs. frozen (R2 = 0.45); frozen vs. frozen drug (R2 =0.9999). Figure 1. Median IC50 Comparison. Median IC50 values are calculated from >3000 samples IC50 values. Percent median is the percent a particular sample IC50 compares to the median of all samples of that disease character. Mean values are shown for inhibitors with replicates. R2 from raw IC50. (A) Comparison of effective drug hits on fresh and frozen primary peripheral blood AML samples (R2 = 0.8151). (B) Comparison of effective drug hits obtained on a freshly isolated AML bone marrow sample versus 2 matched frozen/thawed samples from peripheral blood. Fresh vs. frozen (R2 = 0.45); frozen vs. frozen drug (R2 =0.9999). Disclosures Agarwal: CTI BioPharma: Research Funding. Druker:Oregon Health & Science University: Patents & Royalties; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Sage Bionetworks: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; ARIAD: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.