ALBERT

Description: Several types of tumor cells utilize the phosphatidylinositol 3 Kinase (PI3K)/AKT pathway to support their growth and survival, and blockade of this pathway has been shown to have an antiproliferative effect in these cells. The significance of this survival pathway in Hodgkin disease (HD), and in particular, the neoplastic Hodgkin and Reed-Sternberg (H/RS) of Hodgkin disease (HD) is unknown. We studied routinely processed tissue sections of 42 cases of primary HD (n=42, 35 classical HD and 7 nodular lymphocyte predominant) for the expression of the active phosphorylated form of AKT. We also studied AKT expression in a panel of 4 well-characterized HD-derived cell lines (HD-MyZ, HD-LM2, L-428, and KM-H2). 27 of 42 (64.3%) cases of primary HD sections expressed phospho AKT (23/35 classical and 4/7 nodular lymphocyte predominant). Cultured H/RS cells also showed constitutive phosphorylation of AKT on Ser473. CD30 ligand (CD30L), CD40L and RANKL, increased the phosphorylation of AKT and downstream molecules as early as 1 hour. The effect of 3 small molecule inhibitors of PI3K/AKT pathway (PI3K inhibitor LY294002; AKT inhibitors QLT0394 and QLT0395 from QLT Inc, Vancouver, Canada) on cell proliferation and survival of cultured H/RS cells was examined by the MTS assay. The PI3K inhibitor LY294002 showed antiproliferative activity in a time and dose dependent manner in all cell lines tested. This antiproliferative effect was primarily due to cell cycle arrest in G0/G1 phase, as determined by propidium iodide staining, and to a less extent due to induction of apoptosis, as determined by the Annexin-V binding assay. However, when the AKT inhibitors QLT0394 and QLT0395 were used, they primarily induced apoptosis even at nanomolar doses. Apoptosis was mediated by caspase 8 activation as determined by western blot and was partially reversed by the pancaspase inhibitor ZVAD-FMK. Both AKT inhibitors alteredf AKT phosphorylation within 24 hours, and reduced phosphorylation of downstream molecules, such as 4E-BP1. Moreover, the AKT inhibitor QLT0395 showed downregulation of MDM2 and cyclin D1 within 24 hours, and increased ERK1/2 phosphorylation with subsequent decrease of total ERK1/2 levels at the same time frame, but had no effect in BCL-2, cFLIP or Bax. Importantly, both AKT inhibitors (QLT0394 and QLT0395) maintained their killing activity even in the presene of CD30L, CD40L, and RANKL. These preclinical data suggest that small molecules hitting different targets within PI3K/AKT pathway may have different effects on proliferation and apoptosis and that blockade of this pathway may be of therapeutic value in the treatment of patients with HD.

Description: Survivin, a member of the inhibitors of apoptosis protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignancies, including leukemias. To better understand its role in acute myeloid leukemia (AML), we profiled survivin expression in samples obtained from 511 newly diagnosed AML patients and in CD34+38− AML stem/progenitor cells using a validated reverse-phase protein array; we correlated its levels with clinical outcomes and with levels of other proteins in the same sample set. We found that survivin levels were higher in bone marrow than in paired peripheral blood leukemic cells (n = 140, P = .0001) and that higher survivin levels significantly predicted shorter overall (P = .016) and event-free (P = .023) survival in multivariate Cox model analysis. Importantly, survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk blasts and total CD34+ AML cells (P 〈 .05). Survivin expression correlated with the expressions of multiple proteins involved with cell proliferation and survival. Particularly, its expression strongly correlated with HIF1α in the stem/progenitor cell compartment. These results suggest that survivin is a prognostic biomarker in AML and that survivin, which is overexpressed in AML stem/progenitor cells, remains a potentially important target for leukemia therapy.

Description: We have used Reverse Phase Protein Arrays (RPPA) to perform proteomic profiling in Acute Myelogenous Leukemia (AML) focusing on cell cycle, apoptosis and signal transduction pathway proteins (ASH 2006, abstract #107). Protein expression signatures were derived from this dataset of 436 AML patients, analyzed for 30 total and 22 phopho- proteins. The predictive ability of these RPPA derived protein expression signatures has not been prospectively tested to determine if they are valid. This dataset presented an opportunity to validate this as there was a population of patients with known FLT3-ITD and D835 mutation status (n=297) and another population where the status was unknown (n=139), among which 55 had sufficient sample available for mutation analysis. Prior to performing the mutation analysis a predictive model was built using linear regression with part of the data utilized for training and the reminder for validation. The model was designed to predict for the presence of mutation, either ITD or D835, although there are differences int eh signature of each. The total population had 85 cases with FLT3-ITD and 15 with the D835 mutation. The optimal model that was developed, using 30%, 50% and 70% of the samples for training and the remainder for validation, had a median validation accuracy of 68%, 70% and 72% respectively. Prospective predictions of FLT3-ITD or D835 mutation status were then made for all samples lacking FLT3-ITD or D835 mutation data. Mutation analysis was then performed using PCR amplification followed by 2-D gel electrophoresis (FLT3-ITD) to evaluate for PCR product size, or sequencing (D835) on 55 samples. This revealed 9 cases with FLT3-ITD, 3 with a D835 mutation, 1 with both and 43 without mutation. Among these 55 cases the model correctly predicted that 8 of the 12 mutant cases would be mutant including 8 of 10 with a FLT3-ITD, but 0 of 2 with only the D835 mutation. Among the 43 wildtype cases 36 were accurately predicted to be wildtype, while 7 were incorrectly predicted to have the mutation mutant. This yields an overall accuracy (OA) of 80%, Sensitivity =66%, Specificity=90%, Positive Predictive Value (PPV) of 53%, False positive rate of (FPR) of 16%. Since most patients with FLT3-ITD have Diploid cytogenetics we also looked at the predictive accuracy of the protein expression signature in that population. Among 23 patients with Diploid cytogenetics the overall accuracy was OA) of 83%, Sensitivity =75%, Specificity=87%, Positive Predictive Value (PPV) of 75%, False positive rate of (FPR) of 13%. Since FLT3-ITD and D835 carry different prognostic impact, and had different protein expression signatures, greater accuracy might be achieved if separate models were developed for each mutation individually. The model demonstrated that RPPA derived protein expression signatures can accurately be used to predict mutation status providing the first prospective validation of protein expression signatures in AML.

Description: 1. Serum lactic dehydrogenase (SLD) was measured in 66 patients with leukemia. Elevated levels were observed in 23 (77 per cent) of 30 adults with acute leukemia; in 7 (70 per cent) of 10 children with acute leukemia; in 15 (94 per cent) of 16 patients with chronic myelocytic leukemia, and in 6 (60 per cent) of 10 subjects with chronic lymphatic leukemia. 2. The degree of SLD elevation appeared to be significantly greater in the chronic myelocytic than in the chronic lymphatic group. 3. Serial determinations of SLD were performed for periods of from 1 to 82 weeks in 55 patients, of whom 43 had elevated levels at one time or another. Fair to good correlation between SLD and conventional parameters of leukemic activity was noted in 7 (33 per cent) of adult acute leukemias; in 2 (40 per cent) of child leukemias; in 8 (67 per cent) of chronic myelocytic leukemias; and in 1 (20 per cent) of the chronic lymphatic group. 4. Possible sources of enzymes are discussed. At the present time the mechanism of SLD elevation in leukemia remains an unsolved problem. 5. Serum transaminase (SGOT) levels were studied in 61 subjects. Levels greater than 100 units were seen in only 8 patients, of whom 7 had either viral or toxic hepatitis and 1 had terminal septicemia. Other types of liver impairment, including leukemic infiltration, were associated with normal or slightly elevated SGOT.

Description: Because protein function regulates the phenotypic characteristics of cancer, a functional proteomic classification system could provide important information for pathogenesis and prognosis. With the goal of ultimately developing a proteomic-based classification of acute myeloid leukemia (AML), we assayed leukemia-enriched cells from 256 newly diagnosed AML patients, for 51 total and phosphoproteins from apoptosis, cell-cycle, and signal-transduction pathways, using reverse-phase protein arrays. Expression in matched blood and marrow samples were similar for 44 proteins; another 7 had small fold changes (8%-55%), suggesting that functional proteomics of leukemia-enriched cells in the marrow and periphery are similar. Protein expression patterns were independent of clinical characteristics. However, 24 proteins were significantly different between French-American-British subtypes, defining distinct signatures for each. Expression signatures for AML with cytogenetic abnormalities involving −5 or −7 were similar suggesting mechanistic commonalities. Distinct expression patterns for FMS-like tyrosine kinase 3–internal tandem duplication were also identified. Principal component analysis defined 7 protein signature groups, with prognostic information distinct from cytogenetics that correlated with remission attainment, relapse, and overall survival. In conclusion, protein expression profiling patterns in AML correlate with known morphologic features, cytogenetics, and outcome. Confirmation in independent studies may also provide pathophysiologic insights facilitating triage of patients to emerging targeted therapies.

Description: We have previously demonstrated that the protein expression and activation profile of apoptosis and signal transduction pathway (STP) proteins in acute myelogenous leukemia (AML) cells that survive in vitro chemotherapy exposure (Survivor Cells) differ markedly from that seen in the bulk population of AML blasts present at diagnosis. This methodology, although cumbersome, could provide insight into the adaptive changes utilized by the small fraction of cells that survive chemotherapy but which ultimately lead to relapse and death in the majority of AML patients. Unfortunately, the small number of available survivor cells (24 hr) increases in BCL-XL, BCL2, XIAP, Bax, Bad, pBad112, pBad 136, pBad155, PKCα and pPKCα that did not develop in survivors of 10μM ara-C. When NB4 cells were treated with IDA alone, similar levels and patterns of expression were seen for all proteins, except pBAD-136, when compared to ara-C alone. In contrast, levels and patterns in U937 cells treated with IDA showed lower total AKT and BAD and higher BCL-XL, pERK, XIAP compared to ara-C treated cells. When U937 cells were treated with both agents, the timing and expression levels mirrored that seen in cells treated with IDA alone. NB4 cells treated with both agents showed transient increases in pERK from 30 min to 4 hours and a late increase in pPKCα but were otherwise similar in comparison to cells treated with IDA or ara-C alone. We conclude that the pattern of STP and apoptosis regulatory protein expression and activation in survivor cells does differ over time and varies by the dose and combinations of chemotherapy agents used. Studies in patient-derived samples will be studied to see if these patterns might be utilized to predict sensitivity or resistance to selected therapies.

Description: Abstract 1397 The ERBB family of receptor tyrosine kinases (EGFR, Her-2, Her-3 and Her-4) are receptor tyrosine kinases that, through mutation or aberrant expression, serve as oncogenes by promoting hallmark behaviors of cancer in many solid tumors. Previous work has suggested that HER2 is expressed in as much as 30% of B-ALL patients, and correlates with chemoresistance. We therefore hypothesized that HER2 signaling in Ph+ ALL may augment growth signaling and promote other malignant behaviors, such as resistance to cell death and independence from growth factors. Western blot and flow cytometric analyses of two human Ph+ ALL cell lines, Z119 and Z181, revealed cell surface expression of HER2, but not other family members. To determine the role of HER2 signaling in Ph+ ALL cell lines, the pan-HER family small molecule kinase inhibitor canertinib was used, and reverse phase protein array (RPPA) was conducted in Z119 and Z181 cell lines. Briefly, lysates from canertinib treated cells were spotted using a GeneTAC™ G3 arrayer onto nitrocellulose-coated FAST® slides. Incubation of the slides was performed with forty-three antibodies directed towards various cell signaling proteins followed by colorimetric detection and results were subsequently validated by western blotting. RPPA analyses revealed that treatment with canertinib effectively diminished HER2 phosphorylation in both cell lines. Additionally, we found decreased phosphorylation of the pro-survival molecules ribosomal protein S6, p70S6kinase, and c-Src, as well as increased expression of the pro-apoptotic molecules BIM and cleaved-PARP in both Ph+ ALL cell lines. Congruent with these findings, elevated activity of the executioner caspase 3 and increased DNA fragmentation, two distinct biochemical markers of apoptosis, were present after canertinib treatment in Z181 and Z119 cells, suggesting that inhibition of HER2 signaling results in programmed cell death of Ph+ ALL cell lines. This induction of apoptosis paralleled a decrease in overall proliferation of these cell lines, further implicating HER2 signaling in proliferation of Ph+ ALL. Next, we analyzed if clinically approved inhibitors of HER2 function could be utilized to produce the same biological consequence as canertinib in Ph+ ALL cell lines. Lapatinib (Tykerb) is a dual EGFR/HER2 small molecule kinase inhibitor approved by the FDA for the treatment of breast cancer. Consistent with our results utilizing canertinib, lapatinib was capable of inhibiting proliferation of both Z119 and Z181 cell lines. Interestingly, the FDA approved monoclonal antibody HER2 inhibitor trastuzumab (Herceptin) did not inhibit proliferation of these cell lines. Similarly, trimerized herceptin conjugates, which improve internalization of HER2 receptor, also had no effect on Ph+ ALL cell line proliferation. These results highlight an important distinction between the effects of the intracellular small molecule inhibitors of HER2 and monoclonal HER2 antibodies. In particular, extracellular engagement of the HER2 receptor by monoclonal antibodies may not be effective in targeting the HER2 signaling pathways required for proliferation and survival of Ph+ ALL. Taken together, our studies suggest that HER2 may play an important role in growth and survival signaling of Ph+ ALL cell lines and inhibition of HER2 with small molecule kinase inhibitors may improve treatment regimens. Thus, additional studies are warranted to determine the importance of HER2 in clinical specimens and the potential benefit of combining HER2 inhibitor therapy with imatinib treatment for Ph+ ALL. Disclosures: Mills: Glaxosmithkline: Research Funding; Pfizer: Research Funding.

Description: The β integrins play an important role in the cell-to-cell interactions, which trigger intracellular signal transduction pathways. Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate Akt, which promotes cell survival. On the other hand, PI3K/Akt and JAK/STAT signaling pathways are also recognized as potent anti-apoptotic mediators activated by ligation of growth factor receptors. We have previously demonstrated that stroma cells protect acute promyelocytic leukemic (APL) cells from apoptosis (Tabe, Blood103:1815–1822, 2004). Here, we investigate the ability of bone marrow stroma cells to activate Akt, STAT3, and ILK signaling in leukemic cells co-cultured with stroma in low-serum conditions (0.5% FCS). Human mesenchymal stem cells (MSC), co-cultivated with APL-derived NB4 cells in direct cell-to-cell contact, partially inhibited spontaneous apoptosis and enhanced viability of NB4, while separation from stromal cells by transwell insert abrogated this supporting effect of MSC. Western blot analysis using phosphospecific antibodies demonstrated that direct cell-to-cell contact with MSC caused strong activation of Akt and STAT3 signaling in NB4 cells, which have low baseline phosphorylation of these proteins. Treatment with PI3K inhibitor LY294002 or JAK/STAT3 inhibitor (AG480) decreased both, Akt and STAT3 activation in NB4 cells, however, in cells co-cultured in direct contact with MSC the Akt and STAT3 phosphorylation levels were still significantly higher than in suspension cultures and in cells separated by transwell. These observations indicate cross-talk between PI3K/Akt and JAK/STAT pathways, and that Akt is activated independent from PI3K in NB4 cells through direct interaction with MSC. Both, LY294002 and AG480 induced apoptosis and decreased viability of suspension NB4 cells, but this effect was partially abrogated by MSC co-culture. Next, we examined the effects of these signal transduction inhibitors on MSC. MSC expressed both, phospho-Akt and phospho-Stat3, which was inhibited by LY294002 and AG480. LY294002 but not AG480 induced moderate apoptosis in MSC (annexin V positivity; MSC alone19.7 %; LY294002, 30.8%; AG480, 20.1% at 72 hours). Finally, we investigated Akt and STAT3 activation associated with ILK in NB4 cells. Treatment with ILK inhibitor KP004 (QLT Inc., Vancouver, Canada) decreased phosphorylation of Akt and STAT3 only in NB4 cells co-cultured with MSC and not in suspension cultures. The specific abrogation of MSC-mediated signaling resulted in higher induction of apoptosis in stroma co-cultured cells compared to suspension cells (annexin V positivity; KP004 treated suspension cultures 47.4±4.3%; MSC co-cultures 64.9±10.3%). These results indicate that bone marrow stroma cells support survival of leukemic cells through β integrin linked ILK, which activates Akt in a PI3K-independent manner and also stimulates STAT3. We propose that abrogation of ILK/Akt and STAT3 signaling may overcome protective effects of the bone marrow microenvironment on APL cells and thereby greatly enhance anti-leukemic therapies.

Description: We have previously demonstrated that protein expression profiling assessing the activation state of apoptosis and STP proteins could be successfully performed in Acute Myelogenous Leukemia (AML) patient samples using reverse phase protein arrays (RPPA). With the goal of providing comprehensive proteomic based classification of AML we have now generated an array using protein derived from the leukemia enriched fraction of 559 primary AML samples. Included are 326 samples from 263 newly diagnosed AML patients (176 Marrow, 149 blood samples, 63 with concurrent blood and marrow specimens), 61 primary refractory, and 163 relapse specimens. Also present on the array are 120 purified peptides, 18 cell lines, 18 normal peripheral blood or bone marrow samples. All specimens are printed with 5 serial 1:3 dilutions in duplicate on geographically separate locations of the slide. As a control for printing and staining variations each pair of samples is flanked by a positive control cell line mixture and buffer only negative control. Each array has a total of 8064 dots printed using a Aushon 2470 Arrayer. 64 slides were produced and within 7 days probed with 54 validated antibodies against apoptosis and STP proteins, including 20 phos-specific antibodies (ABs), demonstrating the high throughput capability of the system. Seven duplicate slides were stained a week after the first staining to test reproducibility. Spot intensities were quantified using MicroVigene software. The positive and negative control printed across the slide were used to generate a topographical map of staining density across the slide. The negative control values are subtracted on a regional basis to provide background correction and the cell line positive control is utilized to normalize the range. The purified peptides permit protein quantification. The developed slides generally were of very high quality with all but 3 slides providing interpretable data. Intra- and inter- slide reproducibility was very high. The samples set was randomly divided into a test and validation set. Unsupervised hierarchical clustering analysis on the proteins is being performed using perturbation bootstrap resampling to assess the significance of clusters. Preliminary analysis suggests that several reproducible clusters exist, similar to our findings with the screening array (see Tibes et al., ASH 2005) and that these clusters correlate with remission attainment, relapse or overall survival. This dataset will undergo addition analysis after additional proteins are assayed to yield a more comprehensive proteomic based classification of AML. This sample set contains 123 patients on our cytokine array (see Kornblau et al., ASH 2006) permitting association between cytokine expression level and the activation status of the STP and apoptosis pathways. This array provides the ability to rapidly screen large numbers of AML patient samples for expression and activation state. Distinct pathway activation patterns can classify AML, provide prognostic guidance and may serve to triage patients to emerging targeted therapies aimed at these pathways.

Description: We have previously shown that the protein expression profile and especially the activation state of apoptosis and STP proteins are highly prognostic in AML. The development of RPPA permits a more comprehensive analysis of protein expression and phosphorylation (phos) patterns in the STP and apoptotic cascades. We have generated a screening array using protein derived from the leukemia enriched fraction of 94 primary AML samples (with 21 concurrent blood and marrow specimens), comprised equally of patients that were primary refractory (PrimRef), or that achieved complete remission that was continuous (CCR) or which relapsed after 6 to 24 months (REL). These slides have been probed with 22 total and 15 phos-specific antibodies (ABs) against apoptosis and STP proteins. Spot intensities were quantified using MicroVigene and the data for each protein standardized data by subtracting the mean expression levels across samples and dividing by the standard deviation. Unsupervised hierarchical clustering analysis on the proteins was performed. We found one small cluster of 5 proteins: Cyclin D1, β-catenin, phos-NMP, p53, and AMPK. The remaining proteins formed another cluster, with a subset of 8 proteins somewhat further separated. In several cases (mTOR, JNK, and PTEN) the total protein amounts clustered as nearest neighbors to the phos version of the same protein. Using perturbation bootstrap resampling to assess the significance of clusters we found 4 reproducible clusters and the structure suggests additional clusters in this data set. A Fisher exact test showed that these were significantly associated with response (p = 0.03) and with the prognostic category defined by cytogenetics (p = 0.04) but was not associated with the source (blood vs. marrow) of the sample (p = 0.67). Ten proteins were differentially expressed between cytogenetically defined prognostic groups: total and phospho AKT, PTEN and JNK, p-mTOR, p-STAT3, pp38 and PS6.p24.44 (all with P