Identification of targeted therapies for rare adult mature <scp>T</scp>‐cell leukemia using functional <i>ex vivo</i> screening of primary patient samples
Authors: Uma Borate, Brianna A. Norris, Pierrette Lo, Cristina Tognon, Jeffrey Tyner, Stephen Spurgeon
Journal: American Journal of Hematology (2017)

T-cell prolymphocytic leukemia (T-PLL) is a, rare and aggressive, αβ post-thymic T-cell lymphoproliferative disorder found in patients with a median age of 64 years.1 Introduced in 1997, Alemtuzumab (Campath-1H) improved the median overall survival in T-PLL patients to approximately 20 months from 7.5 months.2 However, only about 60% of patients reach a complete remission (CR) and at least half of these patients relapse within 1 year.2 Those who achieve a CR are encouraged to proceed with allogeneic stem cell transplantation (SCT), however nearly half of SCT patients relapse from their disease.3 Genetic analyses of T-PLL have revealed a number of chromosomal rearrangements and mutations. The most common abnormalities occur in chromosome 14 (75% of cases) with an inv(14)(q11q32), followed by 8q (70% of cases) with isochromosome 8q or additional genetic material.4 Chromosome 14 abnormalities result in the overexpression of TCL1 and AKT, thus enhancing cell proliferation.4 Additional abnormalities include the loss of ataxia telangiectasia mutated (ATM) due to deletions of 11q and a high mutation rate of JAK1, JAK3, and STAT5B.4, 5 It is hypothesized that small molecule inhibitors either directly, or indirectly, targeting the PI3K/AKT and/or JAK-STAT pathways may disrupt overexpression of cells in patients with these mutations.4, 5 However, the lack of research specific to T-PLL makes it difficult to study targeted therapy for this disease. Thus, an ex vivo functional screening platform using multiple small molecule inhibitors simultaneously is an efficient method to investigate therapies that may be effective in patients with this disease. We present the first report retrospectively analyzing primary T-PLL patient samples and assessing their sensitivity against various targeted agents using our ex vivo functional screening platform.6 After obtaining informed consent using an IRB sample collection protocol, bone marrow aspirate and/or peripheral blood were obtained from patients with various hematological malignancies including five patients with T-PLL. We analyzed information from the medical charts of all five patients. The results are found in Figure 1A. The median age was 65 years (range, 49-88 years) at diagnosis. Analysis of karyotype and fluorescent in situ hybridization (FISH) testing showed all patients had a gene inversion (14q11). Patient 3 and 5 both showed complex karyotypes positive for ATM deletion. Analysis of effective drugs screened in five T-PLL patients. A: Demographics and clinical summary of T-PLL patients. B: Our ex vivo assay identifies dasatinib as the most effective small-molecular inhibitor in 4 out of 5 T-PLL patients with JNJ-28312141 being the second most effective. C: The top five most effective drugs screened against T-PLL patients were found to target at least one subunit (either α, β, or both) of the Platelet-derived growth factor receptor (PDGFR). Each drug is ranked 1-5 (1 being the most effective). Other targets overlapping between 80% and 60% of drugs are Vascular endothelial growth factor 2 (VEGFR2) and c-KIT as well as BCR-ABL, respectively. [Color figure can be viewed at wileyonlinelibrary.com] All patients achieved a CR after Campath-1H treatment. One patient underwent SCT in CR1, two patients relapsed with severe disease progression, and two other patients relapsed then proceeded with re-induction. The patients who underwent re-induction proceeded with SCT in CR2. All patients, who underwent SCT, had a median progression free survival (PFS) of 6.37 months (4.5-7.7 months) after SCT. Overall, the median overall survival (OS) for all patients was 19.1 months (5.06-28.0 months). Specifically, patients who underwent SCT in CR1 or CR2 had a median OS of 11.0 months (8.2-20 months). Our study, along with others, re-enforces that despite Campath-1H therapy and SCT in CR1 or 2, patients have a poor outcome, similar to published literature.3, 4 We performed our ex vivo drug screening assay on these patient samples. We hypothesized that small molecular inhibitors targeting AKT/JAK-STAT or other disrupted pathways not yet associated with this disease would show sensitivity in these T-PLL patient samples. Fresh primary cells from each individual patient were isolated using a ficoll-density gradient and simultaneously plated with dose-escalating concentration gradients of 143 single-agent small-molecular inhibitors.6 This included kinase inhibitors (TKIs), bromodomain inhibitors, BHM mimetics, proteasome inhibitors, IDH1/2 inhibitors, and several other drug classes. We calculated the IC50 value using a second-degree polynomial regression curve which fits through 8 data points.6 IC50 values were considered effective if they were less than or equal to fivefold below the median IC50 for all samples tested across all hematological malignancies.6 Nine (6.2%) of the 143 small-molecular inhibitors tested were effective by our assay definition. The effective drugs included: 6 FDA approved TKIs (axitinib, dasatinib, sorafenib, pazopanib, and ponatinib), midostaurin, an NF-kB activation inhibitor, and 2 CSF1R inhibitors. Sensitivity of T-PLL patients to these drugs is shown in Figure 1B. Dasatinib, JNJ-28312141, ponatinib, axitinib, and pazopanib were the five most effective drugs in four out of five of these T-PLL patients. Dasatinib showed the most uniform efficacy with 4 of the 5 patients showing sensitivity, with a median IC50 of 3 nM (range 2-28 nM), followed by JNJ-28312141—a CSF1R inhibitor—with a median IC50 of 24 nM (range 11 nM-6760 nM). Possible, overlapping targets, between the top five most effective drugs were visualized using a Venn diagram in Figure 1C. Platelet-derived growth factor receptors (PDGFR)—with subunits α, β, or both—were targeted by all drugs. In addition, vascular endothelial growth factor 2 (VEGF2) and c-KIT were targeted by 80% of the five most effective drugs, whereas BCR-ABL was targeted by 60% of these agents. PDGFR binding can result in downstream activation of several non-receptor TKIs in the SRC, ABL, AKT, and JAK families.7 Hence, PDGFR and VEGFR inhibitors may indirectly disrupt downstream signaling of JAK-STAT, ABL mutated pathways, or the PI3K/AKT pathway, therefore reducing cell proliferation.7 In conclusion, our ex vivo drug sensitivity assay allows us to analyze the effect of multiple targeted therapies simultaneously in primary patient samples of a rare leukemia that is otherwise difficult to study. Using this assay, we have identified dasatinib—an FDA approved therapy—as a potentially promising treatment for T-PLL. We have also brought to attention the importance of investigating the role of PDGFR, VEGFR, c-KIT, and BCR-ABL in T-PLL as these were all also targets of the most effective drugs in the sensitivity assay. In summary, there is a dire need for new treatments for T-PLL and the development of pathway-specific therapies, in addition to Campath-1H, would substantially improve the overall survival for this disease. Clinical trials using targeted therapies are urgently needed. Aptose Biosciences, Array Biopharma, AstraZeneca, Constellation Pharmaceuticals, Genentech, Incyte, Janssen, Seattle Genetics, Takeda, CTI, Cylene, Lorus, MolecularMD, Agios, Ambit BioSciences, D3 Oncology Solutions, Gilead Sciences, Roche, ARIAD, BMS, Novartis, Oncotide Pharmaceuticals, Dana-Farber Cancer Institute, Array, Curis, and Pfizer