ALBERT

Description: Abstract 2436 The occurrence of ABL-kinase (ABL-K) mutation is a major persisting concern in CML patients treated with tyrosine kinase inhibitor (TKI) therapies. Leukemic stem cell niche can protect the leukemic cells by providing survival and/or quiescence signals but also could favor the occurrence of ABL-kinase mutations. Amongst the ABL-kinase mutations, T315I is one of the most problematic as it induces resistance to all three clinically accepted TKI (Imatinib, Dasatinib, Nilotinib) and has been shown to occur at the level of stem cells (Chomel et al, Leuk lymphoma 2010). Ponatinib (Formerly AP24534) is a multi-targeted TKI which overcomes resistance to T315I mutation as well as to other ABL-kinase mutations. To model the role of the niche in the context of T315I in patients treated with Ponatinib, we designed a niche-based cell mutagenesis assay in the human hematopoietic UT7 cells engineered to express BCR-ABL (UT7-BCR-ABL native cells) and BCR-ABL with T315I (UT7-T315I), via retrovirus mediated gene transfer. Western blot analyses demonstrated that these cells express BCR-ABL and UT7 clones harboring the T315I mutation were resistant to Imatinib, Dasatinib and Nilotinib but sensitive to Ponatinib. We have treated UT7-T315I cells with N-ethyl-N-nitrosourea (ENU, 50microg/ml) for 24 hours and seeded them on previously prepared MS5 stromal cells (plate of 96 wells) in the presence of Ponatinib (30 nM final concentration). As a control, ENU-treated cells were also cultured in the absence of MS5 feeders and Ponatinib-based selection was performed in the same conditions. The same niche-based assay was also performed in the UT7-BCR-ABL native cells and selection process has taken place in the presence of Imatinib (2 microM). Cell medium was changed every week with addition of Ponatinib (UT7-T315I cells) or Imatinib (UT7-BCR-ABL native cells). At week+4, Ponatinib-resistant and IM-resistant wells cultured in the presence (MS5+) or in the absence (MS5-) of the hematopoietic niche were enumerated. In UT7-BCR-ABL native plates, the numbers of IM-resistant outgrowth was identical in MS5+ versus MS5- conditions (51/95 and 54/96, respectively). On the other hand, in UT7-T315I plates, there was a major difference in the numbers of Ponatinib-resistant T315I cells in MS5+ (96/96, 100% survival) as compared to MS5- conditions (65/96, 62 % survival). IM and Ponatinib-resistant clones were amplified and 48 clones resistant to Ponatinib (MS5+ n= 24; MS5- n= 24) and 48 clones resistant to IM (MS5+ n= 24: MS5- n=24) were screened for ABL-K mutations using a denaturing gradient gel electrophoresis assay followed by direct sequencing of the ABL-kinase domain. In the native BCR-ABL mutagenesis assay (with Imatinib), all clones were found mutated, in the presence or absence of MS5 stromal cells. Most of them were mutated in the P-loop region (E255K, G250E, Y253H). In addition, we have found 3 clones harboring the T315I mutation (1 in MS5- and 2 in MS5+ conditions). Concerning T315I clones surviving to Ponatinib, in addition to the T315I mutation, most of the resistant clones harbored an additional P-loop mutation which occrred in the absence or presence of MS5. Moreover, in two clones, two additional mutations were detected in addition to the T315I mutation. Interestingly, when Ponatinib-resistant cells were switched from MS5+ conditions to MS5- conditions, Ponatinib resistance could be abrogated in some but not all cases. In conclusion, our assay shows that the hematopoietic stem cell niche could play a crucial role in conferring resistance to Ponatinib, not only via the occurrence of novel mutations but also by providing survival signals. Preliminary results also suggest that the hematopoietic niche could facilitate the emergence of T315I mutation in cells expressing native BCR-ABL. These results could be important to study the mechanisms of the occurrence and selection of ABL-K mutations in patients treated with TKI including Ponatinib, and to develop niche-targeted therapies to overcome TKI-resistance in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.

Description: Abstract 883 Currently Imatinib Mesylate (IM) represent the first line therapy for chronic myeloid leukemia (CML). Recent data suggest that despite unprecedented rates of complete cytogenetic responses (CCR) and major molecular responses (MMR) obtained, leukemic stem cells (LSC) persist in the majority of patients (pts). LSC have been shown to be resistant to in vitro treatments with tyrosine kinase inhibitors (TKI). Consequently, discontinuation of IM in pts with undetectable molecular residual leukemia (UMRL) attested by RQ-PCR, leads to molecular relapses in the majority of the cases. Although the persistence of CD34+ CD38- leukemic stem cells has been demonstrated in pts with complete cytogenetic remission (CCR), the persistence of BCR-ABL+ leukemic stem cells in UMRL pts with has not been studied so far. For this purpose, we have performed an extensive analysis of bone-marrow-derived clonogenic and primitive hematopoietic stem cells in 6 pts with RQ-PCR constantly negative in their blood samples. Concerning the treatments; 5 out of 6 pts were off therapy, 3 pts (UPN1, 2, 3) had been treated with interferon-a only (IFN) for 6–13 years and their therapy was discontinued for 11, 16 and 8 years, respectively and 2 pts (UPN4 and 5) had been treated successively with IFN and IM and their IM therapy was discontinued for 2 years. One patient (UPN6) had been treated with IM followed by dasatinib and was on dasatinib at the time of the study. UPN7 was previously treated with first IFN then IM (which induced a stable UMRL) and then she switched to dasatinib because of side effect with IM. Bone marrow cells were collected and CD34+ cells purified using immunomagnetic columns. After performing a clonogenic assay, CD34+ cells were used in long-term culture initiating cell (LTC-IC) assays with weekly half medium changes. At week+5, clonogenic assays were performed and LTC-IC-derived clonogenic cells activity was calculated. For each patient 20 individual and 20 pools of 10 clonogenic cells and 20 individual and 20 pools of 10 LTC-IC derived CFU-C were plucked in order to obtain information in at least 220 CFU-C. After RNA extraction, BCR-ABL was quantified by RQ-PCR and in each positive CFU-C a nested PCR was performed to confirm the results. In one patient (UPN7) a NOD/SCID mouse assay was performed. All 3 pts treated with IFN alone, had BCR-ABL+ clonogenic cells varying from 0.5% (UPN1, 2) to 45 % (UPN3). All 3 had LTC-IC derived CFU-C positive for BCR-ABL (UPN1: 20%; UPN2 5%; UPN3 3%). In two pts previously treated with IFN and IM, clonogenic CFU-C BCR-ABL positivity was 10% and 5% whereas LTC-IC-derived CFU-C was 5% in UPN4) and undetected on UPN5. In UPN6 treated with IM then dasatinib, 5% of CFU-C was BCR-ABL+ whereas 100% of LTC-IC derived CFU-C was positive. The analysis of SCID-NOD assays performed in CD34+ cells from patient UPN7 is ongoing. Overall, these data show, for the first time to our knowledge, that in pts in IFN and IFN/IM-induced long-term remissions, there is persistent clonogenic BCR-ABL+ output maintained by BCR-ABL-expressing stem cells in the absence of relapse. In the only patient with successively treated with IM and dasatinib, 100 % of primitive hematopoietic stem cells are BCR-ABL+, despite PCR-negativity in peripheral blood, suggesting their possible quiescence in vivo and highlighting a theoretical risk of relapse. It remains to be determined if in pts with TKI-induced remissions, the analysis of stem cell compartments could be of use for clinical decisions to discontinue therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3365 Recent data suggest that the occurrence of T315I mutation, due in part to genetic instability induced by BCR-ABL, confers to the hematopoietic cells acquiring this mutation an additional genetic instability. Specific signalling pathways induced by T315I mutation could be at the origin of this phenomenon. As T315I substitution arises as a subclone of BCR-ABL-expressing cells, it is very difficult to study the specific effects of the putative mutational effect in primary CML cells. To determine if an additional genetic instability is induced in the presence of T315I, we have used the human UT7 cell model in which we have stably introduced BCR-ABL and BCR-ABL T315I using retroviral vectors. Western blot analysis showed that BCR-ABL protein levels were equivalent in both cell lines. To determine the global genomic instability phenomenon induced by BCR-ABL we have performed a mutator phenotype assay using the detection of HPRT mutants in the presence of 6-Thioguanine (6-TG) in control, wild-type BCR-ABL and BCR-ABL T315I-expressing UT7 cells in methycellulose assays. 6-TG resistant HPRT mutant colonies were quantified 3–4 weeks after initial seeding. The same cells were also used to quantify the baseline levels of &Ugr;-H2AX phosphorylation, indicative of double strand breaks (DSB). In addition, the same experiments were performed after the co-culture of the cells in the presence of a hematopoietic niche. Results: In the experimental conditions used, 6-TG resistant colonies were identified only in T315I-mutated cells, with a frequency of 7.9 10−6. The individually plucked HPRT mutants grew in liquid cultures in the presence of 6-TG, confirming the presence of a 6-TG resistance. Interestingly, the co-culture in the presence of MS-5 cell line for 3 days, increased the mutator phenotype (12. 10−6). The analysis of baseline g-H2AX levels showed a clear increase of &Ugr;-H2AX phosphorylation in UT7-T315I cells as compared to UT7-BCR-ABL which showed slightly higher levels as compared to control. To determine the long-term influence of Imatinib in the mutator phenotype, we cultured T315I cells in the presence of IM 1 microM for 7, 21 and 36 days followed by a mutator assay. The frequency of mutator phenotype was found to be stable, varying from 4.4 10−6 at day 7 to 5.24 10−6 at day 36 in the presence of IM. To determine the influence of the niche on the genomic instability, we have treated the UT7, UT7-BCR-ABL and UT7-T315I cells with mitomycin C (MMC) for 6 and 18 hours in the absence or in the presence of MS5 cells, followed by quantification of &Ugr;-H2AX levels at day+3. in these conditions, MMC induced '-H2AX levels were higher in T315I-mutated cells as compared to UT7 and the co-culture with MS-5 cells reduced this phenomenon. Thus, T315I mutation confers higher levels of DSB in steady state and increases the mutator phenotype of BCR-ABL-expressing cells. The co-culture in the presence of the niche reduces DSB but further increases the frequency of mutator phenotype, suggesting that the niche is not the major factor of genetic instability but stimulates the self-renewal of T315I-mutation bearing leukemic cells. Disclosures: No relevant conflicts of interest to declare.

Description: BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation. Disclosures: Guilhot: Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.

Description: Abstract 4181 Ahi-1 (Abelson helper integration site-1) is an oncogene that was initially identified by provirus insertional mutagenesis in v-abl-induced murine pre-B cell lymphoma. The Ahi-1/AHI-1 protein contains an SH3 domain, multiple SH3 binding sites and a WD40-repeat domain, all known to be important mediators of protein-protein interactions. Human AHI-1 is highly deregulated in human leukemic cells, particularly in BCR-ABL+ leukemic stem cells from patients with chronic myeloid leukemia (CML). We have demonstrated that overexpression of Ahi-1 in primitive hematopoietic cells confers a growth advantage in vitro and induces leukemia in vivo; these effects can be enhanced by BCR-ABL, a fusion oncogene that plays a major role in the genesis of CML. AHI-1 can physically interact with BCR-ABL and JAK2 in CML cells and this interaction complex further mediates tyrosine kinase inhibitor (TKI) response/resistance of CML stem/progenitor cells. Despite its importance, the mechanism by which this complex affects cell proliferation and survival and regulates sensitivity of CML cells to TKIs remains unknown. To identify and characterize which functional domain(s) of Ahi-1 is critical for its interaction with BCR-ABL and/or Jak2, full length Ahi-1 and several mutant forms, including N-terminal deletion (N-terΔ, containing both SH3 and WD40-repeat domains), SH3 deletion (SH3Δ) and double WD40-repeat domain and SH3 domain deletions (SH3WD40Δ) were generated and stably transduced into BCR-ABL inducible BaF3 cells, in which the level of expression of BCR-ABL can be down-regulated by exposure to doxycycline. Epitope-tagged full length and mutant Ahi-1 constructs were also transiently expressed in 293T cells co-expressed with either BCR-ABL or Jak2. Co-IP experiments showed that Ahi-1 is highly expressed and stably associated with BCR-ABL-Jak2 complex in BCR-ABL inducible cells co-transduced with full-length Ahi-1 and that it can directly interact with Jak2; Ahi-1 was detected in both Ahi-1-transduced BaF3 cells (without BCR-ABL) and BCR-ABL inducible cells after IP with a Jak2 antibody. Interestingly, N-terminal region of Ahi-1 was required for Ahi-1-Jak2 interaction as a predicted 70 kD product was not detectable in the same cells transduced with the Ahi-1N-terΔ mutant. In contrast, N-terminal region of Ahi-1 was not associated with Ahi-1-BCR-ABL interaction since BCR-ABL was still detectable in BCR-ABL inducible cells co-transduced with the Ahi-1N-terΔ mutant after IP with the anti-ABL antibody. The deletion of either the SH3 domain or both the SH3 and WD40-repeat domains did not interfere with their interaction with Jak2. Ahi-1 lacking both the SH3 and WD40-repeat domains lost its ability to interact with BCR-ABL when the SH3WD40Δ mutant and BCR-ABL were co-expressed in 293T cells and the SH3 deletion did not affect Ahi-1's interaction with BCR-ABL, indicating that the WD40-repeart domain is required for a direct Ahi-1-BCR-ABL interaction. As expected, overexpression of full-length Ahi-1 in BCR-ABL inducible cells resulted in fewer Annexin V+ apoptotic cells after imatinib (IM) treatment compared to BCR-ABL inducible cells (5.9% and 8% v.s.26% and 34% with either 2 or 5 μM IM) after 24 hours. Interestingly, cells expressing the SH3WD40Δ mutant displayed dramatically increased cell sensitivity to IM with increased Annexin V+ cells (62% and 69% v.s.5.9% and 8%), while cells expressing the SH3Δ and the N-terΔ mutants had similar numbers of Annexin V+ cells as compared to BCR-ABL inducible cells (32% and 16% v.s.26% with 2 μM IM). Colony forming cell assays (CFC) further showed significantly reduced growth factor independent CFC numbers generated from the SH3WD40Δ mutant compared to BCR-ABL inducible cells co-expressed with the full-length Ahi-1 (4% v.s.46% with 5μM IM compared to untreated cells). Interestingly, a gene expression study comparing full-length Ahi-1-transduced BCR-ABL cells with its mutants further demonstrated that these changes were associated with deregulated expression of Bcl-2 and Cis (Cytokine inducible SH2 protein) involved in apoptosis. These results suggest that the WD40-repeat domain is important for Ahi-1's role in mediating IM-induced apoptosis and that targeting this critical domain of Ahi-1may provide a novel therapeutic option for treatment of CML. Disclosures: No relevant conflicts of interest to declare.

Description: Genetic instability is a hallmark of chronic myeloid leukemia (CML). Recently, several major abnormalities in DNA repair mechanisms have been identified in primitive CML cells that likely explain the additional mutations these cells develop leading to their selective growth under tyrosine kinase inhibitor (TKI) therapies. It seems likely that such mechanisms also underlie disease progression in CML. However, an understanding of the specific somatic mutations involved and investigations of their resulting effects on the biological behavior of primary sources of primitive chronic phase (CP) CML cells is extremely challenging. As an alternative approach, we have now explored the possibility of applying whole genome sequencing (WGS) to induced pluripotent stem cells (iPSCs) derived from primitive CML cells to determine if such iPSCs, genocopy the mutations present in the diagnostic sample from which they were generated and whether primitive hematopoietic cells derived from these iPSCs might be useful for future drug screening experiments. To this end, we chosen a CML patient whose CP clonogenic cells contained both the Ph1 chromosome and the JAK2 V617F mutation and whose disease progressed into an accelerated phase (AP) during TKI therapy. iPSC were generated from leukemic cells obtained at the time of AP using Oct4, Sox2, Klf4 and c-Myc gene transfer. The presence of both BCR-ABL and JAK2 V617F was confirmed in 24/24 iPSC colonies. A control iPSC line negative for both genes was similarly established from the patient’s CD34+CD31+ endothelial progenitors purified from peripheral blood. We then performed WGS on DNA prepared from the leukemic cells obtained at diagnosis of CP (CML 006), the AP cell-derived iPSCs (PB34), and the control non-leukemic iPSCs (PB13), using a HighSeq Illumina platform. WGS revealed 845,175 somatic SNVs and 68,817 somatic short Indels in the CP leukemic cells at diagnosis that were not present in the non-leukemic iPSCs (PB13). 49,225 of these SNVs and 11,665 of the short Indels were novel (absent in the dbSNP database), and 419 were found in the COSMIC database. We identified 274 novel SNVs (3 missense, 161 nonsense, 108 synonymous and 2 splice site mutations) and 46 short Indels (19 insertions and 27 deletions). Most of the novel coding SNVs and Indels were heterozygous and an estimation of the variant allele frequency indicated these were present in virtually all leukemic cells. In addition to the JAK2 V617F mutation that was present at diagnosis, we found a novel frame shift mutation in exon 12 of ASXL1 gene (p.S871YfsX5) leading to protein truncation, a genetic event that has also been associated with myeloproliferative neoplasms (MPNs) and AML. We also identified several novel SNVs predicted by SIFT, Provean and PolyPhen-2 algorithms to be deleterious for protein structure. These novel mutations were found in genes relevant for the pathophysiology of MPNs, including the catenin (CTNNA1 R204C, and AIDA K235T), RAS (RREB1 P789T), autophagy (ULK1 R553C) cellular antioxidant defense (GSR S293C), RNA nuclear transport (NUP160 start loss) pathways. Individual sequencing confirmed the presence of these mutations in PB34 and their absence in PB13 (non-leukemic iPSC). We next compared the sequence data from the AP leukemic cell-derived iPSCs (PB34) with the diagnostic data (CML006). This analysis showed only 799 additional somatic SNVs and 96 new short Indels compared with those already evident in the cells present at diagnosis. Only 4 (3 non synonymous and 1 synonymous) SNVs and no Indels were found in exons. These mutations could have appeared during the application of the reprogramming process to the AP leukemic cell-derived iPSCs; none was an obvious contributor to MPN pathophysiology. Finally, we showed that day16 embryoid bodies derived from the PB34 iPSCs contained expected numbers of CD34+ cells (18±11%, n=6) and BCR-ABL-expressing hematopoietic colony-forming cells (CFCs, 143±64 / 105 cells, n= 6). These CFCs showed a slight inhibitory response to imatinib (54±15% colonies obtained in 1 µM IM, n=4) whereas a combination of IM and Pimozide (a STAT5 phosphorylation inhibitor), reduced survival another ∼10-fold. In conclusion, we have provided proof-of-principle results illustrating the potential of iPSC technology in combination with WGS to dissect the clonal evolution of disease progression in CML and develop patient specific drug screens that could build on this data. Disclosures: Turhan: BMS, Novartis: Honoraria, Research Funding.

Description: Abstract 4789 Pluripotency and self-renewal, two key characteristics of induced pluripotent stem cells (IPS), make these cells ideally suited for modeling diseases in vitro and generating biological resources usable for drug screening and cell therapy. However, the reprogramming efficiency of somatic cells greatly varies according to the cell type, to the in vitro proliferation index, the number of passages and the age of the donor. Human amniotic liquid-derived cells (hALDC), collected during amniocentesis for the prenatal diagnosis of genetic diseases, represent an abundant source of primary cells. In preliminary experiments we have shown that hALDC expressed endogenous Oct4 and Sox2 proteins suggesting that could be readily amenable to reprogramming. To this end, we have used two strategies using either hALDC or neonatal fibroblasts: (1) lentivirus mediated gene transfer of OCT4, SOX2, LIN28, NANOG, (2) retroviruses mediated gene transfer of OCT4, SOX2, CMYC, KLF4 and (3) lentiviral transfer of OCT4, SOX2. hALDC transduced by these viruses were placed on MEF and b-FGF (10 ng/ml) with daily medium changes. One to three weeks after infection, typical human ES-like colonies could be picked up for expansion before being characterized. HALDC show an increased reprogramming potential with the [OCT4, SOX2, LIN28, NANOG] and [OCT4, SOX2] cocktails, when compared to reprogramming of neonatal fibroblasts. Twelve hALDC-derived-IPS cells were obtained from 12 different samples of amniotic fluid. All hALDC-IPS cell lines maintained a normal karyotype in culture and displayed the morphology and characteristics of human embryonic stem cells, including the surface expression of Tra-160, SSEA-3, SSEA-4, HESCA-1 and alkaline phosphatase, and formed multi-lineaged teratomas upon injection to NOD-SCID mice. Gene expression profiles of the IPS cell lines reveal a high correlation coefficient between hALDC-iPS cells and human embryonic stem cells, and a low correlation between hALDC-iPS and hALDC. When compared to hES cells H1, H9 and Cl01, these cell lines generated hematopoiesis with a variable efficiency in vitro. Amongst the hALDC-IPS cell lines generated by our laboratory (http://www.hescreg.eu/) four lines carry an inherited trisomy of chromosome 21, and three lines carry the homozygous “S” mutation in the beta-globin gene of sickle-cell anemia. All hALDC-IPS cell are currently banked at the Human Pluripotent Stem Cell Core Facility, France. In conclusion, hALDC can be rapidly and efficiently reprogrammed to pluripotency with a limited number of transgenes. Moreover, hALDC-IPS cell lines derived from patients can be used to modelize in vitro the phenotypic features of monogenic diseases such as sickle cell anemia or more complex, multifactorial disorders such as Down's syndrom. The ability to generate hematopoietic differentiation from these cell lines will facilitate the modelling of these hematopoietic disorders. Disclosures: No relevant conflicts of interest to declare.

Description: The ABL tyrosine kinase inhibitor (TKI) Imatinib Mesylate (IM) is effective at inducing clinical remission in early phase chronic myeloid leukemia (CML) patients, but is not curative. Early relapses and acquired drug resistance remain some issues in IM-treated patients. In particular, relapses are frequently associated with point mutations in the BCR-ABL tyrosine kinase domain (TK, 〉 50%). Newer TKIs, dasatinib (DA) and nilotinib, have increased potency over IM and show a broader spectrum of activity against mutant forms of BCR-ABL. However, none of these agents is able to target a critical T315I gatekeeper mutation of BCR-ABL in TKI-resistant patients. We have demonstrated that CML stem cells are genetically unstable and generate many BCR-ABL TK mutations in vitro and in vivo. They are also less responsive to TKIs and are a critical target population for TKI-resistance. Thus improved treatment approaches to specifically target CML stem cells and BCR-ABL-T315I resistant cells are clearly needed. It has recently been reported that estrogen receptor variant ERα36 is highly deregulated not only in breast cancer cells, but also in liver cancer and leukemic cells, and that targeting this specific variant with a small molecular inhibitor (Icaritin, SNG162) inhibits CML cell growth. However, the underlying molecular mechanisms of these observations are not understood. Whether this inhibitor, alone or in combination with new ABL inhibitors, can target primary CML stem/progenitor cells and T315I-resistant cells have also not been investigated. In this study, we utilized two cell line model systems: K562 cells and IM-resistant K562 cells without BCR-ABL TK mutation and human UT7 cells expressing either wild type BCR-ABL or carrying the BCR-ABL-T315I mutation. We have now demonstrated that protein expression of ERα36 is highly upregulated in both IM-resistant and BCR-ABL-T315I mutant cells as compared to control cells. Interestingly, the use of pre-clinically validated ERα36 inhibitors (SNG162 and SNG1153) alone inhibits cell growth and induces apoptosis of these cells. BCR-ABL-T315I cells are more sensitive to ERα36 inhibitors treatment, with twofold increases in Annexin V+ cells detected in BCR-ABL-T315I cells after SNG1153 treatment, compared to those detected in BCR-ABL expressing cells. These effects can be further enhanced by combination treatment with a TKI. Importantly, we have discovered that treatment of IM-resistant and BCR-ABL-T315I mutant cells with SNG162 and SNG1153 inhibitors, alone or with a TKI, significantly reduced phosphorylation of BCR-ABL on tyrosine residue 177 (Tyr177), a residue essential for BCR-ABL induced leukemogenesis through its binding to GRB2 and activation of the downstream RAS-MAPK pathway. This new observation was supported by detection of a significant reduction in phosphorylation of MEK1/2 kinase, an important component of the RAS-MAPK pathway, in these cells. Most importantly, IP-Western analysis further demonstrated that the BCR-ABL-GRB2 protein interaction was markedly interrupted in cells treated with SNG inhibitors plus a TKI, which correlates with reduced phosphorylation of Tyr177 in these cells. Moreover, colony-forming cell (CFC) assays showed that SNG inhibitors (SNG162 and SNG1153) in combination with a TKI are more effective at inhibiting growth of CD34+ treatment-naïve IM-nonresponder cells as compared to single drug treatment (46% vs. 25%). We further demonstrated that SNG162 and SNG1153 (up to 10 μM and 5 uM) are not toxic to CD34+ normal bone marrow cells. Interestingly, a combination treatment of DA plus SNG1153 dramatically reduced the level of engrafted leukemic cells in transplanted immunodeficient NSG mice and prolonged the survival of these mice compared to mice treated with DA alone. Median survival of DA treated mice is 87.5 days, while some DA and SNG1153 treated mice remain alive for more than 102 days after transplantation. ERα36 thus emerges as an attractive druggable target for combination therapies to target TKI-insensitive CML stem/progenitor cells and T315I-resistant cells. Disclosures Zhang: Shenogen Pharma Group Ltd: Employment. Ding:Shenogen Pharma Group Ltd: Employment. Lin:Shenogen Pharma Group Ltd: Employment. Meng:Shenogen Pharma Group Ltd: Employment.

Description: The use of tyrosine kinase inhibitor (TKI) therapies has dramatically changed the prognosis of chronic myeloid leukemia (CML) and modified the natural history of the disease. However, the generation of ABL-kinase domain mutations due to the genetic instability of leukemic cells continues to be very significant challenge, especially in advanced phases of the disease. Amongst these mutations, T315I is the most problematic as it reduces the binding of Imatinib, Dasatinib or Nilotinib to their target, leading to a total resistance to all three TKIs. Although the mechanism of the appearance of this mutation is not completely known, it is possible that it induces a signalling pathway not entirely similar to that induced by classical wild-type BCR-ABL. From the mechanistic point of view, the mechanism of resistance of T315I-mutated BCR-ABL to Imatinib is related to the substitution of Isoleucine to Threonine, which normally forms a hydrogen bond with Imatinib, this bond being absolutely required for binding of the drugs to the catalytic site. T315I mutation can be identified by direct sequencing, D-HPLC, allele specific PCR or double gradient gel electrophoresis of the PCR products amplified from the BCR-ABL kinase domain. However these techniques cannot identify directly the leukemic cells expressing T315I-mutated BCR-ABL which are known to co-exist with native BCR-ABL-expressing cells in bone marrow. Infrared microspectroscopy allows the identification of metabolic features of mammalian cells, based on their protein, lipid, amid and nucleic acid contents, generating a spectral signature. In this work we asked whether infrared microspectroscopy can be used to identify metabolic changes occurring in single cells bearing BCR-ABL T315I mutation. For this purpose we have used human (UT7) and murine embryonic stem cells (GS2) expressing either native (N) or T315I-mutated BCR-ABL. In the human UT7 cells expressing N-BCR-ABL, we were able to clearly distinguish at the single cell level, cells expressing BCR-ABL from wild type cells using principal component analysis. However, the presence of T315I mutation induced a clearly different signature, allowing a highly significant separation by the use of spectral signature. To confirm the specificity of this signature, we have used UT7 cells engineered to express either native or T315I-mutated BCR-ABL under the control of Doxycycline (DOX) -sensitive promoters. In this TET-OFF system, the addition of doxycycline to the culture medium inhibits BCR-ABL expression as monitored by Western blots, in approximately 6 days. Using this system, at day 0, UT7 cells expressing N-BCR-ABL and T315I mutated BCR-ABL were clearly distinguishable from parental UT7 cells. Upon inhibition of BCR-ABL expression by addition of Doxycycline, the spectral signature of N-BCR-ABL and T315I-mutated BCR-ABL cells became similar at day 2 and indistinguishable at day 4, but still distinguishable from wild type UT7 cells expressing DOX-inducible GFP. To confirm these results in a different cell context we have used a murine embryonic stem (ES) cell line (GS2) which was transduced with either N-BCR-ABL or T315I-mutated BCR-ABL-expressing lentiviral vectors. BCR-ABL-expressing individual clones grown were analyzed to analyze the spectral signature. In this cell line also, infrared microspectroscopy was able to distinguish ES cells expressing BCR-ABL T315I as compared to N-BCR-ABL. Thus, these results suggest that T315I mutation clearly induces metabolic changes different from N-BCR-ABL in leukemic cells, rendering them identifiable by the analysis of nucleic acid, lipid, amid and sugar contents. This new methodology can now be applied to the identification of primary CML leukemic cells harboring T315I at the single cell level and could be of interest for rapid identification of leukemic cells in a single step. This technique can also be used for rapid screening of novel compounds active against T315I mutation using microfluidic technologies leading to novel drug discoveries. Disclosures Turhan: Bristol Myers Squibb, Novartis: Honoraria, Research Funding.

Description: Abstract 908 There is growing evidence that the bone marrow microenvironment could participate to the progression of chronic myeloid leukemia (CML). Recent data show indeed that placental growth factor (PGF) expression is highly induced in stromal cells from CML patients although they are not part of the leukemic clone as they are Ph1-negative (Schmidt et al, Cancer Cell 2011). It is possible that leukemic cells instruct the niche components via extracellular or contact signals, transforming progressively the “normal niche” into a functionally “abnormal niche” by inducing aberrant gene expression in these cells, similar to the pattern that has been identified in cancer-associated fibroblasts (CAF). In an effort to identify the differential gene expression pattern in the CML niche, we have undertaken two strategies of gene expression profiling using a Taqman Low Density Arrays (TLDA) protocol designed for 93 genes involved in antioxidant pathways (GPX, PRDX, SOD families), stromal cell biology (Collagen, clusterin, FGF, DHH), stem cell self-renewal (Bmi1, MITF, Sox2) and hematopoietic malignancies (c-Kit, hTERT, Dicer, beta-catenin, FOXO3). The first strategy consisted in the analysis of mesenchymal stem cells (MSCs) isolated from the bone marrow of newly diagnosed CP-CML patients (n=11). As a control, we have used MSCs isolated from the bone marrow of age-matched donors (n=3). MSCs were isolated by culturing 6–8.106 bone marrow mononuclear cells in the presence of b-FGF (1 ng/ml). At 2–3 weeks, cells were characterized by the expression of cell surface markers (CD105+, CD90+) and by their potential of differentiation towards osteoblastic, chondrocytic and adipocytic lineages. The second strategy aimed to study the potential instructive influence of leukemic cells in the gene expression program of normal MSC after co-culture with either the UT7 cell line expressing BCR-ABL (3 days) or with CD34+ cells isolated from CP-CML at diagnosis (5 days) as compared to co-culture with cord blood CD34+ cells. After culture, CD45-negative MSC were cell-sorted and analyzed by TLDA. All results were analyzed using the StatMiner software. Results: TLDA analysis of gene expression pattern of MSC from CML patients (n=11) as compared to normal MSCs (n=3) identified 6 genes significantly over-expressed in CML-MSC: PDPN (10-Fold Increase), V-CAM and MITF (∼8 Fold increase), MET, FOXO3 and BMP-1 (∼ 5 Fold increase). To confirm these results we have performed Q-RT-PCR in a cohort of CML-MSC (n= 14, including the 11 patients as analyzed in TLDA) as compared to normal MSC. High levels of PDPN (Podoplanin, ∼8 fold increase), MITF (Microphtalmia Associated Transcription factor, 4-Fold) and VCAM (Vascular Cell Adhesion Protein, 2 fold increase) mRNA were again observed on CML MSCs. Our second strategy (co-culture of normal MSC with BCR-ABL-expressing UT7) revealed an increase of IL-8 and TNFR mRNA expression in co-cultured MSCs (∼5-fold ) whereas there was a major decrease in the expression of DHH (∼ 25-fold) upon contact with BCR-ABL-expressing cells. No modification of the expression of PDPN, MITF or VCAM was noted in normal MSC after this 3-day co-culture strategy using UT7-BCR-ABL cells. Current experiments are underway to determine if primary CD34+ cells from CML patients at diagnosis could induce a specific gene expression pattern in normal MSC after 5 days of co-culture. PDPN is a glycoprotein involved in cell migration and adhesion, acting downstream of SRC. It has been shown to promote tumor formation and progression in solid tumor models and is highly expressed in CAFs. MITF is a bHLH transcription factor involved in the survival of melanocyte stem cells and metastatic melanoma. Finally, high VCAM1 mRNA expression by MSCs from CML patients could be involved in increased angiogenesis known to be present on CML microenvironment. In conclusion, our results demonstrate an abnormal expression pattern of 3 important genes (PDPN, MITF and VCAM1) in MSC isolated in CP-CML patients at diagnosis. The mechanisms leading to an increased mRNA expression (instructive or not instructive by leukemic cells) and their relevance to CML biology are under evaluation. Our results, confirming previous data, suggest strongly the existence of a molecular cross-talk between leukemic cells and the leukemic niche. The elucidation of such aberrant pathways in the microenvironment could lead to the development of “niche-targeted” therapies in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.