ALBERT

Description: Bortezomib (BTZ) was recently evaluated in a randomized Phase 3 clinical trial which compared standard chemotherapy (cytarabine, daunorubicin, etoposide; ADE) to standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia. While the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefitting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. We measured total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) in leukemic cells from 483 pediatric patients using Reverse Phase Protein Arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared to CD34+ non-malignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs. 67% for low-HSF1-pSer326 treated with ADEB (P=0.019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and non-phosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs. those with wild type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients that benefit from BTZ-containing chemotherapy.

Description: Abstract 1490 Background: The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML). In AML, p53 mutations have been associated with poor risk cytogenetics (i.e. complex karyotype, −5/−7). However, the function of p53 can also be compromised by protein stabilization and/or expression. The implications of p53 protein expression have not been studied in AML. Methodology: We assessed p53 expression by high-throughput reverse phase protein array (RPPA) technology in 511 pts (719 samples). Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1: 2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status was determined by Sanger sequencing of exons 5 through 9 of the p53 gene. Results: Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). Cases of AML-M3 and –M6 exhibited higher expression of p53 than other FAB subtypes. p53 expression directly correlated with age (p=0.01) and CD34 (p=0.001) and inversely correlated with WBC (p=0.007), BM (p=0.0001) and PB (p=0.0001) blasts, platelets (p=0.007), HLA-DR (p=0.01), CD19 (p=0.02), and survival (p=0.01). High p53 (p53high) expression level was more associated with unfavorable cytogenetics than with favorable or intermediate cytogenetics (p=0.00001). When all cytogenetic abnormalities were considered, pts with −5 had the highest levels of p53 (p=0.00001). Pts with RAS mutations, but not those with FLT3-ITD, NPM1, or IDH1/2, had lower levels of p53 protein. When pts were divided according to the level of p53 protein expression p53high was associated with lower complete remission (CR) rates (51% vs 56%; p=??) and higher relapsed rates (82% vs 62%; p=??). The median overall survival (OS) of pts with p53high and p53low were 29.8 vs. 51 wks (p=0.009). Most cases with p53high had unfavorable cytogenetics and the effect on OS was predominantly seen in that subpopulation with p53high and p53low pts living a medina of 23.4 vs. 36 wks (p=0.07), respectively. In order to determine whether the poor outcomes associated with p53high were due to the presence of a higher rate of p53 mutations among pts with p53high, we determined the p53 mutational status of 55 pts. p53high was highly correlated with the presence of p53 mutations as the latter were detected in 17/40 pts with p53high but in only 1/16 pts with p53low. Importantly, the presence of p53high, both in the presence (29 wks) or in the absence (24 wks) of p53 mutations, was associated with significantly worse overall survival compared with pts with p53low (56 wks; p=0.05, Figure 1). Multivariate analysis indicated that p53 is a significant independent risk factor for survival in AML. The final model included: age (p=0.000001), favorable cytogenetics (0.01), unfavorable cytogenetics (p=0.00001), WBC (p=0.0005), albumin (p=0.0003), FLT3-ITD (P=0.04), and P53 (P=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), c-MET (p=0.01), FoxO3a (p=0.004), KIT (p=0.001), p38p180p182 (p0.02), BAD (p=0.0001), cleaved PARP (p=0.002), cleaved PARP (p=0.01), TCF4 (p=0.02), fibronectin (p=0.02), and hsp70 (p=0.003), and negatively with AKTp473 (p=0.01), ERK (p=0.002), mTOR (p=0.005), PI3Kp85 (p=0.002), PKCδ (p=0.00002), GAB2 (p=0.00005), beclin (p=0.007), JMJD6 (p=0.001), Gata3 (p=0.02), p21 (p=0.01), and Mdm2 (p=0.001). Conclusions: Our results suggest that high levels of p53 protein constitute a powerful marker of short survival in AML. This effect is independent of p53 mutational status. The poor outcome of pts with high level of expression of p53 in the absence of p53 mutations suggests that the p53 pathway may be functionally perturbed in a much higher proportion of pts with AML than previously recognized. These data support the use of p53 protein expression levels in prognostication and in the development of targeted therapeutics. Disclosures: No relevant conflicts of interest to declare.

Description: Deregulation of signal transduction pathways (STPs) may promote leukemogenesis by conferring cell proliferation and survival advantages in acute myelogenous leukemia (AML). Several agents targeting STPs are under development; however, redundancy and cross-talk between STPs could activate multiple downstream effectors and this could negate the effect of single-target inhibition. The frequency of concurrent activation of multiple STPs in AML and the prognostic relevance of STP activation in AML are unknown. STP protein expression (PKCα, ERK2, pERK2, AKT, and pAKT) was measured by Western blot in samples from 188 patients with newly diagnosed, untreated AML. In univariate and multivariate analysis high levels of PKCα, ERK, pERK, and pAKT, but not AKT, were adverse factors for survival as was the combination variable PKCα-ERK2&pERK2-pAKT. Survival progressively decreased as the number of activated pathways increased. Patients were more likely to have none or all 3 pathways activated than was predicted based on the frequency of individual pathway activation, strongly suggesting that cross-activation occurred. Simultaneous activation of multiple STPs is common in AML and has a progressively worse adverse effect on prognosis. It is thus likely that only combinations of agents that target the multiply activated STPs will be beneficial for patients with AML.

Description: Background The highly evolutionarily conserved Hippo pathway (HP) regulates proliferation, apoptosis polarity and stem cell maintenance. In Humans, TAZ (transcriptional co-activator with PDZ-binding motif) and YAP (Yes associated protein) are the functional effectors, regulating gene expression by co-activating several transcription factors (RUNX, TEADS, SMADS). Phosphorylation inactivates both, causing expulsion from the nucleus and either proteosomal degradation or sequestration by 14-3-3 or membrane protein complexes. A 4 protein complex of LATS 1/2 kinase, MST1/2, MOB and SAV1, phosphorylates YAP and TAZ, and is regulated by many pathways including Neurofibromin 2 (NF2) , and WNT, and by cell-cell contact. YAP and TAZ share many binding and interaction partners, sharing overlapping functions, but they also have exclusive interactions leading to some exclusive functions, notably in SMAD family and TGFβ regulation. Deregulation of HP is associated with numerous solid tumors (breast, lung, colorectal, ovary, liver), with upregulation conferring an adverse prognosis, but mutations of HP components is rare, suggesting other mechanism for deregulation. The role of the HP pathway in AML is undefined and similar to most solid tumors, mutations in AML were not observed by TCGA. Methods To define the role of the HP in AML we used reverse phase protein array (RPPA) technology to print protein from leukemia enriched cells from 511 newly diagnosed AML patients. The RPPA was probed with 231 antibodies, including antibodies against both total and phosphorylated HP components NF2, YAP (p-Serine 127) and TAZ (p-Serine 89). We could not validate antibodies for LATS 1 or 2. Results Levels of total NP2, TAZ YAP were often above that of normal CD34+ cells in 30%, 15% and 17% of cases. Levels of phosphorylated NF2 (p=0.07, 23%) and YAP (p=0.009, 10%) were often above, and those of pTAZ (p= 0.008, 29%) below that of normal CD34+ cells. Higher levels of pTAZ were associated with FAB MO and M1 subtypes, higher CD7 and CD34 expression and higher % marrow blasts. Higher levels of pYAP were associated with lower % marrow and peripheral blood blasts and with cases transformed from RAEBT and CMML. The optimum number of principal components for these 6 antibodies was two, depending on the phosphorylation state of YAP and TAZ. Another statistical method determined the optimal number of patient groups to be 6 (figure 1) with expression 1) Pan Off (19% n=79, Yellow in Fig.) 2) High Total (2%, n =8, Dark Pink.) with very high levels of NF2, TAZ and YAP and high pNF2, 3) Active HP (30% n=123, Blue), with moderate total levels but scant phosphorylation, 4) YAP inactive (19%, n=78, Light Pink) and 5) TAZ inactive (22.5%, n=93, Purple) where only one is phosphorylated and 6) Both inactivated (9% n=37, Green) with both heavily phosphorylated. The TAZ inactive motif was prognostically adverse for overall survival (OS) (Median 30 weeks p=0.009) compared to active, pYAP, both inactivated (median 48-60 weeks), while the High Total pattern had longer OS (median 224 wks). Notably, high pTAZ alone or with pYAP was associated with inferior response and OS in patients treated with demethylating and histone deacetylating drugs. Favorable cytogenetics patients with active HP, High pTAZ or both inactive, treated with fludarabine-HDAC had shorter OS and significantly shorter remission duration (52-70 weeks vs. not reached). High pTAZ was associated with higher SMAD family levels and with unfavorable cytogenetics as well as with higher levels of phosphorylated (inactivated) β-Catenin, likely due to cytoplasmic localization. Conclusions HP proteins are heterogeneously expressed in AML and key effectors YAP and TAZ are differentially phosphorylated. Differential phosphorylation of YAP and TAZ has not been previously reported. This suggests that active YAP in the presence of inactive TAZ is inducing expression of genes that provide proliferative or survival advantages to leukemic blasts. The observation that the Highly active HP group did very well may be identifying a high proliferative group that is more susceptible to cycle specific chemotherapy, similar to Burkitt's leukemia. In summary, this data suggest that the HP pathway is active in AML and has a previously undescribed pattern of separate phosphorylation of YAP and TAZ. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3230 Background. Having previously shown that protein expression signatures (ProExpSig), based on the activation state of cell cycle, apoptosis and signal transduction (STP) regulating proteins, existed and were prognostic in AML, we extended this to evaluate protein expression patterns in ALL. Methodology. We have generated RPPA using protein derived from the leukemia-enriched fraction of 283 primary ALL samples from 216 cases with the goal of providing comprehensive proteomic based classification of ALL. Included are 253 samples from 194 newly diagnosed ALL patients (179 Marrow (BM), 95 blood (PB) samples, 41 with concurrent PB and BM specimens), and 14 relapse (REL) samples from these same cases. Phenotypes included 163 pre-B, 22 T-Cell and 9 Burkitt's cases. There were 42 PH1+ cases but only the 28 treated with TKIs were included for outcome analysis. All protein preps were made from fresh cells on the day of collection as we previously observed that protein expression changes dramatically in ALLs cells with cryopreservation. Present as controls were 16 CD34+ BM and 9 normal PB lymphocyte samples. Samples are printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Each array has a total of 6912 dots printed. Slides were probed with 128 antibodies (ABs) against apoptosis, cell cycle, signaling (STP), regulating proteins, integrins, phosphatases etc including 90 vs. total protein, 33 vs. phos-specific sites and 5 vs. caspase or PARP cleavage sites. Spot intensities were quantified using MicroVigene software. Data was analysed using R, with loading control and topographical background normalization being utilized. Results. Proteins were clustered using absolute Pearson correlation and Ward linkage. Based on the Gap statistic there appeared to be 15 constellations of protein with correlated expression. Many formed expected associations e.g., cleaved caspases, phosphorylated STATs, activated STPs. Based on the combination of expression of these 15 constellations, 7 distinct ProExpSig were defined containing between 18 and 37 cases. Phenotype was highly unbalanced with T cell and Burkitt's cases significantly overrepresented and B cell underrepresented in Signature 3 (O/E 5.7,4.2 and .3) and T cell ALL underrepresented in signatures 2, 4,5 and 6 (O/E .47, .76, .4 and .28). PH+ disease was strongly associated with signatures 5 (18 of 22) and 2 (11 of 375 cases). The PH+ dominated Sig5 was characterized by high expression of Myc, ARC, Survivin as well as high levels of activated STPs and Stats. The Ph+ cases in Sig 2 and 5 differed in that Sig5 had high expression of phospho PKCα, GAB2 and Stat 3 and 6, while Sig 2 had higher expression of proteins in the mTOR and TSC2 axis Almost all t(4:11) cases (9 of 11) were in Sig6, which was characterized by high levels of expression of most proteins excluding those that were highly expressed in the PH+ cases. Cases with Burkitt's were characterized by high expression of cyclins D1 and D3, pRB and by high levels of cleaved caspases. The WBC was markedly higher in Sig 3 and 6. The vast majority n=174) achieved remission with 9 primary refractory and 10 induction deaths occurring, so a signature predicting primary resistance was not observed. It was readily evident that there was a group of favorable signatures (1,2, 4 and 7) and another distinctly unfavorable group of signatures (3, 5, and 6). The favorable group had superior remission duration (median 172 vs. 96 weeks, p= 0.35), event free survival (median 132 vs. 72 weeks, p=0.03) and overall survival (median 180 vs. 93 weeks, P=0.04) compared to the unfavorable signatures. Conclusions. ALL is characterized by 7 distinct protein expression signatures which form a favorable and an unfavorable prognostic group. The adverse ProExpSig are associated with significantly shorter remission duration and survival. Differences within these ProExpSig can be used to direct the rational application of various targeted therapies to those patients most likely to benefit from them thereby improving outcome. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2355 The interaction of bone marrow (BM) mesenchymal stem cells (MSC) and acute myeloid leukemia (AML) cells creates a microenvironment (McrEnv) that supports and regulates the survival and proliferation of leukemic cells. These same BM McrEnv interactions can also create a sanctuary that protects subpopulations of AML blasts from chemotherapy. The mechanisms by which the BM-MSC McrEnv effects these changes remain unclear and the heterogeneity of these effects across different subtypes of AML (FAB, WHO or cytogenetics) is unknown. Furthermore, the functional differences between normal BM-MSC and AML BM-MSC biology are undefined. To address this we set out to perform proteomic profiling comparing normal and AML BM derived-MSC and to ascertain how AML BM-MSC protein expression patterns correlated with AML blast protein expression. We cultured BM samples for 4 to 8 weeks in MEM-alpha media with 20% fetal calf serum to isolate AML-MSC (n=106), and NL-MSC (n=70). Cells defined as MSC were positive for CD90 and CD105 and negative for CD45 by flow cytometry. Whole cell protein lysates were prepared. Matched AML protein samples prepared from mononuclear cell fractions from the same BM collection were available for most cases (n=96). We generated a custom Reverse Phase Protein Array (RPPA) from these samples and probed the array with 151 validated antibodies. Statistical analysis was performed by two-way ANOVA using Tukey's test to identify differential expression of individual proteins between the samples and Ingenuity pathway analysis was performed to elucidate differential pathway utilization. Comparison of AML-BM-MSC to NL-BM-MSC demonstrated similar levels of expression for 66 proteins (43.7%) but 85, 67 and 28 were different at the p-value of 〈 0.05, 〈 0.01 and

Description: Background The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML), particularly in cases with high-risk cytogenetics. It has been shown that p53 stabilization, which frequently occurs when the protein is mutated, can compromise its function. We have shown that p53 stabilization, regardless of the presence of mutations, suggesting alterations of other components in the p53 pathway. Methodology p53 expression was determined using high-throughput reverse phase protein array (RPPA) technology in 719 samples from 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status of p53 alleles was assessed by Sanger sequencing of exons 5 through 9. Expression of components of the p53 pathway was determined using standard immunohistochemical techniques. Nutlin-3a was used in in vitro culture experiments. Results Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). p53 expression correlated directly with CD34 (p=0.001) and inversely correlated with WBC (p=0.007), PB and BM blast burden (p=0.0001), and survival (p=0.01). High p53 (p53high) expression was more associated with unfavorable cytogenetics, particularly -5 (p=0.00001). p53high resulted in lower complete remission (CR) rates (51% vs 56%; p=??), higher relapsed rates (82% vs 62%; p=??), and shorter median overall survival (OS; 29.8 vs. 51 wks, p=0.009) compared to p53low pts. Most cases with p53high had unfavorable cytogenetics. We next correlated p53 stabilization with the presence of p53 mutations in 68 pts. p53 mutations were detected in 20/54 (37%) p53high pts and in 0/14 (0%) pts with p53low. p53high, either in the presence (29 wks) or in the absence (24 wks) of p53 mutations (p=1.0), was associated with significantly shorter OS compared with p53low pts (56 wks; p=0.05). Multivariate analysis revealed p53 expression to be an independent risk factor for survival in AML (p=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), BAD (p=0.0001), cleaved PARP (p=0.002), and cleaved PARP (p=0.01), and negatively with p21 (p=0.01), and MDM2 (p=0.001).Given the similar OS in p53high pts carrying mutant or wild-type p53, we scored the immunohistochemical expression of MDM2, MDM4, and p21 in 30 p53high pts (9 p53 mutated, 21 wild-type p53). Overexpression of MDM2 was observed in 44% vs 48% pts with mutant vs wild-type p53, respectively, whereas rates were 67% vs 62% for MDM4, and 0% vs 19% for p21, for each respective genotype. Overall, of the 21 p53high pts carrying wild-type p53, 15 (71%) had overexpression of MDM2 and/or MDM4, whereas 81% had no p21 expression, indicating deficient activation of the p53 pathway similar to those cases carrying mutant p53. We are currently assessing response to nutlin-3a therapy in 24 primary AML samples (4 mutant p53, 20 wild-type p53). Results showing the impact of p53 mutation and/or stabilization, and expression levels of MDM2, MDM4, and p21 on nutlin-3a therapy will be presented. Conclusions p53 stabilization (p53high) is a powerful predictive and prognostic factor in AML, which is independent of the presence of mutant p53 alleles. Poor outcomes in pts with p53high lacking p53 mutations are very frequently associated with overexpression of negative regulators of p53 such as MDM2 and/or MDM4 and p21 downregulation, indicating a functionally altered p53 pathway. These findings may have implications for therapies targeting the MDM2/p53 axis in AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2704 Background. Mutations of NPM1, FLT3 and RAS are common in AML occurring in ∼ 30%, 25% and 10% of cases. The incidence and prognostic implications of the combinations of wildtype (WT) and mutant (MU) NPM1 and FLT3 are well described and have different prognostic implications. How these mutations effect the cellular biology of leukemic blasts at the protein level is unknown as is the effect of a RAS mutation on these combinations. Methods. To understand the biology underlying these changes we utilized a Reverse Phase Protein Array (RPPA) to measure the protein expression of 195 different antibodies in cases of newly diagnosed AML with different combinations of NPM1, FLT3 and RAS mutations. We also examined the MDACC experience with these events in newly diagnosed AML (non APL) patients that were treated at MDACC. Results. NPM1-MU was present in 20% (4/461) including 36% of diploid (67/185) cases and for all or diploid cases was associated with slightly better overall survival (OS, p= 0.07 and 0.15) and significantly better Event Free Survival (EFS, P=0.002 and 0.001) and longer remission duration (RemDur, p=0.0009, 0.001). FLT3-ITD or D835 mutations were present in 22% (99/450) of cases and 35% (65/183) of diploid cases, in which it was associated with inferior OS (p= 0.05 for all and diploid). RAS-MU were found in 16% (64/403) of cases and was not predictive of OS (p=.49) or RemDur (p=0.62). Mutation in both NPM1 and FLT occurred in 9.3% of cases, more commonly than expected by chance (O/E =2.14, Χ2= 39, P