Shann-Ching Chen

Targetable Kinase-Activating Lesions in Ph-like Acute Lymphoblastic Leukemia

BACKGROUND Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

Genetic Alterations Activating Kinase and Cytokine Receptor Signaling in High-Risk Acute Lymphoblastic Leukemia

Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2, and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2-negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy.

Interleukin-7 receptor mutants initiate early T cell precursor leukemia in murine thymocyte progenitors with multipotent potential

Two interleukin-7 receptor mutants identified in human early T cell precursor leukemia are sufficient to induce disease in mice when expressed in primitive, Arf-null thymocytes.

Loss of oncogenic Notch1 with resistance to a PI3K inhibitor in T-cell leukaemia

Mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K)/Akt signalling are prevalent in T-cell acute lymphoblastic leukaemia (T-ALL), and often coexist. Here we show that the PI3K inhibitor GDC-0941 is active against primary T-ALLs from wild-type and KrasG12D mice, and addition of the MEK inhibitor PD0325901 increases its efficacy. Mice invariably relapsed after treatment with drug-resistant clones, most of which unexpectedly had reduced levels of activated Notch1 protein, downregulated many Notch1 target genes, and exhibited cross-resistance to γ-secretase inhibitors. Multiple resistant primary T-ALLs that emerged in vivo did not contain somatic Notch1 mutations present in the parental leukaemia. Importantly, resistant clones upregulated PI3K signalling. Consistent with these data, inhibiting Notch1 activated the PI3K pathway, providing a likely mechanism for selection against oncogenic Notch1 signalling. These studies validate PI3K as a therapeutic target in T-ALL and raise the unexpected possibility that dual inhibition of PI3K and Notch1 signalling could promote drug resistance in T-ALL.

Functional evidence implicating chromosome 7q22 haploinsufficiency in myelodysplastic syndrome pathogenesis

Chromosome 7 deletions are highly prevalent in myelodysplastic syndrome (MDS) and likely contribute to aberrant growth through haploinsufficiency. We generated mice with a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22 and show that mutant hematopoietic cells exhibit cardinal features of MDS. Specifically, the long-term hematopoietic stem cell (HSC) compartment is expanded in 5A3+/del mice, and the distribution of myeloid progenitors is altered. 5A3+/del HSCs are defective for lymphoid repopulating potential and show a myeloid lineage output bias. These cell autonomous abnormalities are exacerbated by physiologic aging and upon serial transplantation. The 5A3 deletion partially rescues defective repopulation in Gata2 mutant mice. 5A3+/del hematopoietic cells exhibit decreased expression of oxidative phosphorylation genes, increased levels of reactive oxygen species, and perturbed oxygen consumption. These studies provide the first functional data linking 7q22 deletions to MDS pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.07839.001

Haploinsufficient loss of multiple 5q genes may fine-tune Wnt signaling in del(5q) therapy-related myeloid neoplasms.

To the editor: Wnt signaling in hematopoietic cells and the bone marrow microenvironment plays a critical role in maintaining the pool of hematopoietic stem cells (HSCs) and in regulating differentiation.[1][1],[2][2] Wnt signaling is tightly regulated by the interplay of multiple cytoplasmic

Abstract 4870: Integrated genomic analysis of hypodiploid acute lymphoblastic leukemia

Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive form of leukemia characterized by multiple whole chromosomal losses and very dismal outcome. Our previous genome wide study of hypodiploid childhood ALL cases treated by the Children9s Oncology Group and St Jude, employed interrogation of DNA copy number alterations using Affymetrix SNP 6.0 microarrays, candidate gene resequencing and gene expression profiling using Affymetrix U133 Plus 2.0 microarrays. These analyses showed that this disease can be divided into multiple subtypes characterized by variation in the degree of aneuploidy, distinct submicroscopic deletions, sequence mutations and gene expression profile. Near haploid ALL (24-31 chromosomes) frequently harbors alterations of genes regulating Ras signaling (67.6%; NF1, NRAS, KRAS, PTPN11, FLT3, and PAG1), IKZF3 (encoding the lymphoid transcription factor AIOLOS; 13.2%), and a histone gene cluster at 6p22 (17.6%), while low hypodiploid ALL (32-39 chromosomes) is enriched for IKZF2 (HELIOS; 52.9%), TP53 (70.6%) and RB1 (41.2%) alterations. A striking finding was exclusivity of Ras signaling and IKZF2/3 alterations, and biochemical indications of Ras pathway activation in both near haploid and low hypodiploid ALL. To further interrogate the genomic changes of hypodiploid ALL, we performed next generation sequencing using either Illumina GAIIx or HiSeq3000 sequencers on both tumor and matched remission DNA. Whole genome sequencing to at least 30 fold haploid coverage was performed on 10 near haploid and 8 low hypodiploid cases, and whole exome sequencing (Agilent SureSelect Human All Exon 50Mb) on 5 near haploid and 1 low hypodiploid cases. The burden of single nucleotide variations (SNVs) and insertion/deletion (indel) mutations was in general low in this ALL subtype, with 0-5 indels and 9-95 SNVs in coding regions and untranslated leader regions in the whole genome sequenced cases, where the majority of cases had fewer than 30 SNVs. Further, the number of structural variations, including the ones too small to be identified by SNP microarray analysis, and structural rearrangements, were also low, with less than 25 structural variations identified in the whole genome sequenced cases. For the whole exomes, between 10 and 42 non-silent SNVs and 1-2 indels were identified per case. No recurrent alterations not previously identified in the hypodiploid cohort were found in these 24 cases, indicating that the initial genome wide study of this cohort identified the major recurrent alterations in hypodiploid ALL. However, the recurrence screening including the remaining 78 near haploid and low hypodiploid cases in our cohort on the alterations identified by the whole genome and exome sequencing study is ongoing. Altogether, these findings provide critical new insights into the genetic basis of hypodiploid ALL, and indicate that therapeutic targeting of the Ras pathway should be pursued in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4870. doi:1538-7445.AM2012-4870

Abstract 3083: The genetic landscape of Ph-like acute lymphoblastic leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BCR-ABL1-like, or “Ph-like” B-progenitor acute lymphoblastic leukemia (B-ALL) constitutes up to 15% of childhood and 30% of adult ALL, and is characterized by a gene expression profile similar to BCR-ABL1 ALL, alteration of IKZF1, and poor outcome. A pilot next-generation sequencing study identified kinase activating alterations in 15 Ph-like ALL cases. The goals of this study were to define the genomic landscape of Ph-like ALL in children and young adults, and to examine the utility of tyrosine kinase inhibitors (TKIs) in patients harboring genetic alterations activating kinase signaling. We studied 1665 B-ALL cases, including 309 childhood standard risk (10.8% Ph-like), 826 childhood high risk (14% Ph-like), 370 adolescent (16-21 years, 21% Ph-like) and 160 young adult (21-39 years; 26% Ph-like) cases. Approximately 50% of Ph-like cases harbored a CRLF2 rearrangement (IGH-CRLF2 or P2RY8-CRLF2). Next-generation sequencing was performed for 160 non-CRLF2 expressing Ph-like cases, including mRNA-seq (141 cases), whole genome sequencing (30 cases) and/or exome sequencing (12 cases). Fusion transcripts were identified using CICERO, a novel mRNA-seq assembly-based structural variation detection method. Over 100 chimeric in-frame fusions were identified, including 29 involving 12 tyrosine kinase or cytokine receptor genes, 15 of which were recurrent: JAK2 (10 partners), ABL1 (6), ABL2 (3), PDGFRB (3), CSF1R, TYK2, NTRK3, PTK2B, IL2RB (1 partner each), and rearrangements of EPOR into the IGH and IGK loci. Together, these rearrangements were present in ∼30% of Ph-like ALL cases. Additional sequence and structural alterations activating kinase signaling were identified in ∼10% of cases (e.g IL7R, FLT3, SH2B3). Despite the diversity of kinase alterations, the majority are predicted to respond to a limited number of TKIs, but many are novel or have not been tested in suitable preclinical models of ALL. We show that expression of RCSD1-ABL1, RANBP2-ABL1, ZMIZ1-ABL1, RCSD1-ABL2, SSBP2-CFS1R and PAX5-JAK2 in Ba/F3 and primary mouse pre-B cultures induces cytokine-independent proliferation and constitutive activation of JAK/STAT signaling. Furthermore, the ABL1, ABL2 and CSF1R fusions were sensitive to dasatinib (IC50 range 1-2nM), whilst PAX5-JAK2 only responded to the JAK2 inhibitor, ruxolitinib. Notably, we show efficacy of dasatinib (20mg/kg/day p.o) in a xenograft model of ETV6-ABL1, with reduction of circulating human CD45+ cells (17.4 vs 88.2%; p<0.0001) and spleen weight (117 vs 321mg; p<0.0001) in dasatinib treated mice (n=5) compared to vehicle treated mice (n=5). These data define the genomic landscape of Ph-like ALL and show that the majority of cases harbor genetic alterations that activate a limited number of kinase signaling pathways. These results provide the basis for prospective precision medicine clinical trials that identify and direct patients with Ph-like ALL to logical TKI therapy. Citation Format: Kathryn G. Roberts, Yongjin Li, Debbie Payne-Turner, Jinghui Zhang, Richard C. Harvey, Yung-Li Yang, Guangchun Song, Jing Ma, Shann-Ching Chen, Jinjun Cheng, Natalia Santiago-Morales, Ilaria Iacobucci, Meenakshi Devidas, I-Ming Chen, Shalini Reshmi, Michael Rusch, Pankaj Gupta, Naomi J. Winick, William L. Carroll, Nyla A. Heerema, Andrew J. Carroll, Elizabeth A. Raetz, Guido Marcucci, Clara D. Bloomfield, Wendy Stock, Steven M. Kornblau, Elisabeth Paietta, Ching-Hon Pui, Sima Jeha, James Downing, Daniela S. Gerhard, Julie M. Gastier-Foster, Mignon L. Loh, Cheryl Willman, Stephen P. Hunger, Charles G. Mullighan. The genetic landscape of Ph-like acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3083. doi:10.1158/1538-7445.AM2014-3083

Abstract 4448: Notch1 inactivation contributes to PI3Ki resistance in T-ALL.

Tyrosine kinases inhibitors induce genotype-specific regression in advanced human cancers. However, these responses are almost invariably transient and patients ultimately relapse due to outgrowth of drug-resistant clones. Thus, increased knowledge of the mechanisms underlying de novo and acquired resistance is essential for enhancing clinical efficacy. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer for which current therapies are toxic and frequently ineffective. Mutations in genes encoding components of the Notch1 and Ras/PI3K/PTEN/Akt signaling networks are prevalent in T-ALL and frequently coexist in individual patients. Thus, inhibiting these pathways alone and in combination is a rational therapeutic strategy. Notch1 inhibition with a γ-secretase inhibitor (GSI) was recently evaluated in refractory T-ALL, with disappointing results that may have been due to dose-limiting gastrointestinal toxicity. We are not aware of clinical trials of Ras/PI3K/Akt/mTOR pathway inhibitors in T-ALL. Retroviral insertional mutagenesis (RIM) in mice is a robust strategy for modeling both the clonal heterogeneity that exists within individual tumors and the genetic diversity found across a population of patients with the same cancer. We generated transplantable T-ALLs by performing RIM in mice expressing oncogenic KrasG12D from its endogenous genetic locus (Mx1-Cre, KrasG12D) and in wild-type (WT) littermates. These aggressive multi-step cancers are characterized by recurring viral integrations in known oncogenes such as Ikzf1 and late occurring acquired Notch1 PEST domain mutations. We have extensively investigated patterns of response and resistance to targeted inhibitors of PI3K (PI3Ki) and MEK (MEKi) in this system. Whereas KrasWT T-ALLs respond to either PI3Ki or to a combination regimen that also include a MEKi, KrasG12D T-ALLs are largely refractory to PI3Ki, but are sensitive to the MEKi + PI3Ki combination. T-ALLs invariably relapsed despite ongoing treatment, and we verified phenotypic drug resistance in relapse leukemias by transplanting and retreating them in vivo. We unexpectedly identified multiple independent PI3Ki resistant T-ALLs with markedly reduced levels of activated Notch1, including some in which Notch1 mutations that were present at enrollment were absent at relapse. Expression of activated Notch1 increases the sensitivity of mouse and human T-ALL cells to a PI3Ki, and loss of activated Notch promotes resistance and enhances PI3K signaling. Thus, oncogenic Notch1 mutations that promote outgrowth during transformation unexpectedly “switch” to become deleterious during in vivo treatment with a PI3Ki. These data suggest that aberrant Ras/PI3K signaling plays a dominant role in T lineage leukemogenesis and also raise the unexpected possibility that administering Notch1 and PI3K inhibitors sequentially or in combination might inadvertently promote resistance. Citation Format: Monique Dail, Jason Wong, Daniel O9Connor, Joy Nakitandwe, Shann-Ching Chen, Jessica Lawrence, James R. Downing, Deepak Sampath, Kevin Shannon. Notch1 inactivation contributes to PI3Ki resistance in T-ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4448. doi:10.1158/1538-7445.AM2013-4448

Abstract P5-09-13: HDAC inhibition re-sensitizes acquired hormone resistant cells to the cytotoxic effect of tamoxifen

Breast cancer still continues to be a major cause of cancer related deaths in women, second only to lung cancer. Administering anti-estrogens and aromatase inhibitors, both in the adjuvant and metastatic settings, in estrogen receptor (ER) positive disease, is one of the most effective treatment strategies. However, prolonged exposure to these drugs leads to the emergence of resistance in about 40% of those who initially respond. Hence, the emphasis of a multitude of studies is to understand the underlying mechanisms of this acquired resistance and implementing means to overcome it. Recent studies have implicated epigenetic modulation of gene expression in the development of resistance to the anti-estrogen tamoxifen. A clinical trial conducted by our group has demonstrated the efficacy of combining the HDAC inhibitor vorinostat with tamoxifen in patients who had progressed on prior anti-estrogen therapies and showed a total 40% clinical benefit (19% objective response and 21% stable disease for more than 6 months). In an effort to identify patients most likely to benefit from this novel therapy, we sought to elucidate the mechanism behind the clinical efficacy of this combination. To this end, we have generated an in vitro tamoxifen resistant cell line (TAMR) by long-term exposure of MCF7 cells to 4-hydroxy tamoxifen. Significantly reduced anti-proliferative effect of tamoxifen and other anti-estrogens in TAMR compared to the sensitive MCF7 cells demonstrates the establishment of resistance to anti-estrogens in this cell line. In TAMR cells, addition of an HDAC inhibitor reverts resistance to tamoxifen. Although estrogen receptor (ER) expression in TAMR cells appears unaltered, the classical genomic signaling of ER in these resistant cells is suppressed and unresponsive to ligands, as deduced from transcription of ER response genes and luciferase assay of an ERE-luciferase construct. However, the ER remains important, since siRNA mediated depletion of ER inhibits cell growth in TAMR cells. Treatment with the dual EGFR/Her2 kinase inhibitor or an AKT inhibitor significantly inhibits the growth of these cells only when combined with tamoxifen, indicating the importance of crosstalk between pathways. Furthermore, Akt and mTOR protein and activities are down regulated by HDAC inhibition, which are further reduced when combined with tamoxifen. SiRNA mediated depletion of ER leads to reduced Akt and mTOR activities which suggests that ER may act as a driver, possibly through its non-genomic function at the plasma membrane activating members of the growth factor signaling pathways. Studies are ongoing to further characterize the interaction of HDAC and ER inhibition on these signaling pathways and to determine their significance to resistance. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-13.