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.

RNAi screen for rapid therapeutic target identification in leukemia patients.

Targeted therapy has vastly improved outcomes in certain types of cancer. Extension of this paradigm across a broad spectrum of malignancies will require an efficient method to determine the molecular vulnerabilities of cancerous cells. Improvements in sequencing technology will soon enable high-throughput sequencing of entire genomes of cancer patients; however, determining the relevance of identified sequence variants will require complementary functional analyses. Here, we report an RNAi-assisted protein target identification (RAPID) technology that individually assesses targeting of each member of the tyrosine kinase gene family. We demonstrate that RAPID screening of primary leukemia cells from 30 patients identifies targets that are critical to survival of the malignant cells from 10 of these individuals. We identify known, activating mutations in JAK2 and K-RAS, as well as patient-specific sensitivity to down-regulation of FLT1, CSF1R, PDGFR, ROR1, EPHA4/5, JAK1/3, LMTK3, LYN, FYN, PTK2B, and N-RAS. We also describe a previously undescribed, somatic, activating mutation in the thrombopoietin receptor that is sensitive to down-stream pharmacologic inhibition. Hence, the RAPID technique can quickly identify molecular vulnerabilities in malignant cells. Combination of this technique with whole-genome sequencing will represent an ideal tool for oncogenic target identification such that specific therapies can be matched with individual patients.

Determining the rise in BCR-ABL RNA that optimally predicts a kinase domain mutation in patients with chronic myeloid leukemia on imatinib.

In imatinib-treated chronic myeloid leukemia (CML), secondary drug resistance is often caused by mutations in the BCR-ABL kinase domain (KD). As alternative therapies are available for imatinib resistance, early identification of mutations may prevent disease progression. Because most patients are routinely monitored by BCR-ABL quantitative polymerase chain reaction (PCR), it is important to define the optimal increase in BCR-ABL that should trigger mutation testing. Expert panels have provisionally recommended a 10-fold BCR-ABL increase as the trigger for mutation screening, acknowledging the lack of consensus. To address this question, we monitored 150 CML patients by quantitative PCR and DNA sequencing. Thirty-five different mutations were identified in 53 patients, and, during 22.5 months (median) of follow-up after sequencing, mutations were significantly predictive of shorter progression-free survival. An unbiased receiver operating characteristic analysis identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). The 2.6-fold threshold approximated the analytic precision limit of our PCR assay. In contrast, transcript rise cutoffs of 5-fold or greater had poor diagnostic sensitivity and no significant association with mutations. We conclude that the currently recommended 10-fold threshold to trigger mutation screening is insensitive and not universally applicable.

High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients.

Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 "noncanonical" point mutations in 7 patients: N-Ras(G60E), K-Ras(V14I), K-Ras(T74P), and K-Ras(A146T). All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

Absence of SKP2 expression attenuates BCR-ABL-induced myeloproliferative disease.

BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF(SKP2) (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G(1) arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2(V617F), and TEL-PDGFRbeta, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2(-/-) marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2(+/+) marrow. SKP2(-/-) leukemic cells demonstrated higher levels of nuclear p27 than SKP2(+/+) counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF(SKP2) may be therapeutically useful.

A half-log increase in BCR-ABL RNA predicts a higher risk of relapse in patients with chronic myeloid leukemia with an imatinib-induced complete cytogenetic response.

PURPOSE: Imatinib induces a complete cytogenetic response (CCR) in most chronic myeloid leukemia patients in chronic phase. Although CCR is usually durable, a minority of patients relapse. Biomarkers capable of predicting those CCR patients with a higher risk of relapse would improve therapeutic management. EXPERIMENTAL DESIGN: To assess whether changes in BCR-ABL RNA levels are a prognostic biomarker predictive of relapse, we regularly monitored transcript levels [every 3 months (median)] in 90 patients with CCR during 49 months (median) of imatinib therapy. RESULTS: Throughout follow-up, the 20 patients with eventual relapse had higher transcript levels than the durable responders. Major molecular response (MMR; >3-log reduction of BCR-ABL RNA) was attained by 76 patients (12 with subsequent relapse) and was a significant predictor of prolonged relapse-free survival (P = 0.0008). A minimal 0.5-log increase in transcripts (before relapse; experienced by 42 patients, 16 with subsequent relapse) conveyed a significantly shorter relapse-free survival (P = 0.0017). Loss of MMR (transcript increase to <2.5-log reduction, before relapse; experienced by 33 patients, 11 with subsequent relapse) was also predictive of shortened relapse-free survival (P = 0.0003). A complete molecular response (undetectable transcripts by nested PCR) was attained by 28 MMR patients (one with subsequent relapse) and conveyed a significantly prolonged relapse-free survival (P = 0.0052). CONCLUSIONS: In chronic myeloid leukemia patients with an imatinib-induced CCR, a minimal half-log increase in BCR-ABL RNA (including loss of MMR) is a significant risk factor for future relapse. The achievement of a complete molecular response imparts longer progression-free survival than the achievement of an MMR.

RNAi screening of the tyrosine kinome identifies therapeutic targets in acute myeloid leukemia.

Despite vast improvements in our understanding of cancer genetics, a large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of numerous types of cancer, but identification and validation of tyrosine kinase targets in cancer can be a time-consuming process. We report the establishment of an efficient, functional screening assay using RNAi technology to directly assess and compare the effect of individually targeting each member of the tyrosine kinase family. We demonstrate that siRNA screening can identify tyrosine kinase targets containing activating mutations in Janus kinase (JAK) 3 (A572V) in CMK cells and c-KIT (V560G) in HMC1.1 cells. In addition, this assay identifies targets that do not contain mutations, such as JAK1 and the focal adhesion kinases (FAK), that are crucial to the survival of the cancer cells. This technique, with additional development, might eventually offer the potential to match specific therapies with individual patients based on a functional assay.

Comparison of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of drug combinations.

BMS-354825 (dasatinib) and AMN107 (nilotinib) are potent alternate Abl inhibitors with activity against many imatinib mesylate-resistant BCR-ABL kinase domain (KD) mutants, except T315I. We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-p210(BCR-ABL) cells to compare incidence and types of KD mutants emerging in the presence of imatinib mesylate, dasatinib, and nilotinib, alone and in dual combinations. Although ENU is expected to induce mutations in multiple proteins, resistant clones were almost exclusively BCR-ABL KD mutant at relevant concentrations of nilotinib and dasatinib, consistent with a central role of KD mutations for resistance to these drugs. Twenty different mutations were identified with imatinib mesylate, 10 with nilotinib (including only 1 novel mutation, E292V) and 9 with dasatinib. At intermediate drug levels the spectrum narrowed to F317V and T315I for dasatinib and Y253H, E255V, and T315I for nilotinib. Thus, cross-resistance is limited to T315I, which is also the only mutant isolated at drug concentrations equivalent to maximal achievable plasma trough levels. With drug combinations maximal suppression of resistant clone outgrowth was achieved at lower concentrations compared with single agents, suggesting that such combinations may be equipotent to higher-dose single agents. However, sequencing uniformly revealed T315I, consistent with the need for a T315I inhibitor, to completely block resistance.

BCR-ABL kinase domain mutations in chronic myeloid leukemia: not quite enough to cause resistance to imatinib therapy?

Patients with chronic myeloid leukemia (CML) treated with imatinib in early chronic phase tend to have durable remissions, but there is a high rate of relapse in patients with advanced disease. Mutations in the kinase domain of BCR-ABL that impair drug binding have been identified as the major mechanism of resistance. It is not known when exactly these mutations arise, but in some patients retrospective analysis of pretherapeutic samples demonstrated identical mutations, suggesting selection in the presence of drug. In the present study we have used a highly sensitive PCR assay to screen for kinase domain mutations in pretherapeutic samples from CML patients, irrespective of their subsequent response to imatinib. We find that kinase domain mutations are demonstrable in approximately 1/3 of patients with accelerated phase or blast crisis and that the presence of two copies of the Philadelphia chromosome is strongly correlated with mutation detection. Unexpectedly, kinase domain mutant clones were not invariably selected in the presence of drug, suggesting that additional mechanisms must contribute to a fully drug resistant leukemia.

High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy.

Mutations in the kinase domain (KD) of BCR-ABL are the leading cause of acquired imatinib resistance. In some cases, identical mutations were detected at relapse and in pretherapeutic specimens, consistent with selection of resistant clones in the presence of drug. However, the incidence of KD mutations in imatinibnaive patients, irrespective of response to therapy, is unknown. We studied mutation frequency in 66 patients with chronic myelogenous leukemia (CML), using cDNA sequencing and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) assays for 8 common mutations. Thirteen patients were positive by ASO-PCR only, 1 by ASO-PCR and sequencing, and 1 by sequencing only (overall frequency, 22.7%). T315I was most frequent (12% of patients). Eleven of the 14 patients with positive ASO-PCR had follow-up samples available for sequencing. Wild-type sequence was detected in 6 of 11, 2 different mutations in 1 of 11, and identical mutations in 4 of 11 patients, 2 of whom had achieved major cytogenetic response. In multivariate analysis mutation detection was associated with clonal cytogenetic evolution, exposure to 6-Thioguanine, and a low platelet count, but not with response to imatinib, event-free survival, and overall survival. KD mutants present at low levels do not invariably lead to relapse, and additional factors are required to induce a fully drug-resistant phenotype.

The JAK2V617F activating mutation occurs in chronic myelomonocytic leukemia and acute myeloid leukemia, but not in acute lymphoblastic leukemia or chronic lymphocytic leukemia.

Activating mutations in tyrosine kinases have been identified in hematopoietic and nonhematopoietic malignancies. Recently, we and others identified a single recurrent somatic activating mutation (JAK2V617F) in the Janus kinase 2 (JAK2) tyrosine kinase in the myeloproliferative disorders (MPDs) polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. We used direct sequence analysis to determine if the JAK2V617F mutation was present in acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML)/atypical chronic myelogenous leukemia (aCML), myelodysplastic syndrome (MDS), B-lineage acute lymphoblastic leukemia (ALL), T-cell ALL, and chronic lymphocytic leukemia (CLL). Analysis of 222 patients with AML identified JAK2V617F mutations in 4 patients with AML, 3 of whom had a preceding MPD. JAK2V617F mutations were identified in 9 (7.8%) of 116 CMML/a CML samples, and in 2 (4.2%) of 48 MDS samples. We did not identify the JAK2V617F disease allele in B-lineage ALL (n = 83), T-cell ALL (n = 93), or CLL (n = 45). These data indicate that the JAK2V617F allele is present in acute and chronic myeloid malignancies but not in lymphoid malignancies.