Relapsed ALL Research Articles

Concurrent Use of Blinatumomab and Donor Lymphocyte Infusion (DLI) for Treatment of Relapsed Precursor B Cell Acute Lymphoblastic Leukemia (pre B ALL) after Allogeneic Hematopoietic Cell Transplant (HCT)

Adults with relapsed pre B ALL after allogeneic HCT historically have poor prognosis, but new therapies may improve outcomes. Blinatumomab is a bispecific engager anti-CD19/CD3 antibody that simultaneously binds cytotoxic T cells and CD19+ normal and malignant B cells. Each cycle consists of a 4-week continuous infusion. Blinatumomab engages endogenous T cells and is usually used as a bridge to HCT; however in the post-HCT setting donor T cells are engaged. DLI used for treatment of ALL relapse after transplant exerts modest effect, and we hypothesized that co-administration of blinatumomab and DLI may have synergistic anti-leukemic activity. We report 2 cases of relapsed Philadelphia chromosome (Ph) negative pre B ALL after HCT treated concurrently with blinatumomab and DLI. Case 1: A 35 year-old woman with pre B ALL received a matched related donor peripheral blood HCT in first remission using clofarabine and busulfan preparative regimen. Marrow relapse occurred 136 days after HCT with 24% donor chimerism. She received salvage chemotherapy with 2 DLI (5 x 107 CD3 cells/kg each) but had persistent disease (7% marrow blasts and 99% donor chimerism). Blinatumomab was subsequently started. After 2 cycles marrow showed complete hematologic remission, 98% donor chimerism and persistent cytogenetic abnormality. Third and fourth DLI were given on day 15 of the third and fourth cycles of blinatumomab. No adverse effects were noted during lymphocyte infusion. There was no exacerbation of the mild chronic skin GVHD present prior to the third cycle of blinatumomab. Extramedullary relapse in a CD19+ sacral mass was discovered 1 month after cycle 4 of blinatumomab, successfully treated with radiation. Patient currently has second extramedullary ALL relapse with a CD19- lung mass (16 months after HCT and 11 months after first relapse) with no ALL in the marrow and 100% donor chimerism. Case 2: A 71 year-old woman with Ph-negative pre B ALL received reduced intensity conditioning matched unrelated donor peripheral blood HCT in second remission using fludarabine, 2 Gy TBI and ATG as preparative regimen. She developed flow cytometry and cytogenetic relapse 106 days after HCT with 97% donor chimerism. Blinatumomab was started and DLI (1 x 107 CD3 cells/kg) was given on day 2 of cycle 3 of blinatumomab without adverse events. She developed grade 3 late onset acute skin and gut GVHD 15 days after completion of cycle 3 of blinatumomab which was successfully treated with corticosteroids. No further cycles of blinatumomab or DLI were given and she remains in complete remission for 6 months. Co-administration of DLI and blinatumomab appears to be safe with no acute adverse effects observed in 2 patients. Dual therapy resulted in anti-leukemia effect in both cases of pre B ALL relapsed after HCT. A larger series is needed for further investigation of the safety and efficacy of this approach.

The combination effects of bendamustine with antimetabolites against childhood acute lymphoblastic leukemia cells.

Bendamustine combined with other drugs is clinically efficacious for some adult lymphoid malignancies, but to date there are no reports of the use of such combinatorial approaches in pediatric patients. We investigated the in vitro activity of bendamustine combined with other antimetabolite drugs on B cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines established from pediatric patients with refractory or relapsed ALL. We also developed a mathematically drown improved isobologram method to assess the data objectively. Three BCP-ALL cell lines; YCUB-2, YCUB-5, and YCUB-6, were simultaneously exposed to various concentrations of bendamustine and cladribine, cytarabine, fludarabine, or clofarabine. Cell growth inhibition was determined using the WST-8 assay. Combinatorial effects were estimated using our improved isobologram method with IC80 (drug concentration corresponding to 80 % of maximum inhibition). Bendamustine alone inhibited ALL cell growth with mean IC80 values of 11.30-18.90 μg/ml. Combinations of bendamustine with other drugs produced the following effects: (1) cladribine; synergistic-to-additive on all cell lines; (2) cytarabine; synergistic-to-additive on YCUB-5 and YCUB-6, and synergistic-to-antagonistic on YCUB-2; (3) fludarabine; additive-to-antagonistic on YCUB-5, and synergistic-to-antagonistic on YCUB-2 and YCUB-6; (4) clofarabine; additive-to-antagonistic on all cell lines. Flow cytometric analysis also showed the combination effects of bendamustine and cladribine. Bendamustine/cladribine or bendamustine/cytarabine may thus represent a promising combination for salvage treatment in childhood ALL.

IFITM3 (CD225) Links the B Cell Antigen CD19 to PI3K-AKT Signaling in Human ALL Cells

Background & Hypothesis: IFITM3 (Interferon-induced transmembrane protein 3) also known as CD225/Leu-13 was identified as interferon-inducible molecule in the context of viral infection. IFITM3 encodes a surface receptor, basally expressed on the plasma membrane, that is associated with known B cell co-receptors including CD19, CD81 and CD21. Recent immunotherapy approaches based on CD19-specific engineered chimeric antigen receptors (CART19) have achieved spectacular successes in eliminating pre-B ALL cells based on surface expression of CD19 (Grupp et al., 2013). However, in some cases, CART19 treatment was followed by ALL relapse developing from clones that lacked CD19 surface expression. Therefore, we studied factors that regulate CD19 surface expression on human pre-B ALL cells.Results: Studying IFITM3 mRNA levels in ALL cells at the time of diagnosis in clinical trials for childhood (COG P9906) and adult ALL (ECOG E2993), we found that higher than median expression levels of IFITM3 predicted shorter overall and relapse-free survival (P=0.014). In addition, patients with higher than median IFITM3 mRNA levels at the time of diagnosis were significantly more likely to experience ALL relapse and had a higher risk of a positive MRD status at the end of induction chemotherapy.To study the function of Ifitm3 in a model for human pre-B ALL, pre-B cells from Ifitm3-/- mice were transformed with BCR-ABL1. Strikingly, deletion of IFITM3 resulted in loss of CD19 expression on the surface of normal and leukemic pre-B cells. Besides loss of surface expression, loss of Ifitm3 also caused impaired phosphorylation of CD19-Y513, which mediates downstream activation of PI3K-AKT signaling in both B cell progenitors and pre-B ALL cells. These changes were paralleled by G0/1 cell cycle arrest (P<0.001), loss of colony formation capacity (P=0.0004) and activation of checkpoint molecules p53 and p21, reduction of BCL2 and BCLXL levels and increased propensity to apoptosis. Reconstitution of IFITM3 reversed these changes.Conversely, forced expression of IFITM3 in patient-derived pre-B ALL cells increased phosphorylation of CD19-Y513 together with downstream SRC, SYK, PI3K signaling. Although human IFITM1 has known as a component of B cell receptor complex, co-immunoprecipitation experiments revealed that the cytoplasmic tail of IFITM3 interacts with CD19, LYN, SYK, PI3K p110δ and AKT. In addition, agonistic antibodies against IFITM3/CD225 trigger CD19/PI3K-AKT signaling, which caused massively increased proliferation of pre-B ALL cells. Interestingly, agonistic antibodies against CD19 had no effect on the proliferation of pre-B ALL cells despite their ability to activate CD19/PI3K-AKT signaling. These findings indicate a specific role of IFITM3 in regulating CD19/PI3K-AKT signaling in malignant pre-B ALL cells compared to their normal pre-B cell counterparts.Conclusion: These findings identify novel role of the IFITM3 (CD255) surface receptor in regulating CD19 phosphorylation and CD19-Y513 mediated PI3K-AKT signaling in pre-B ALL cells. IFITM3 is a central mediator of CD19 surface trafficking and mediates the proliferation and survival signaling via PI3K-AKT in normal pre-B cells and various subtypes of human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance.Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors.In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008).Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. DisclosuresNo relevant conflicts of interest to declare.

Identification of BCL6 As a Therapeutic Target in RAS-Driven Acute Lymphoblastic Leukemia

▪Background & Hypothesis: In ~50% of cases of acute lymphoblastic leukemia, activating lesions in RAS pathway are found. These lesions are particularly frequent in relapse ALL and specifically acquired in the relapse clone (Irving et al., 2013). A previous study of senescence rescue screens identified the transcriptional repressor BCL6 as a key factor to overcome p53 dependent senescence and enable RAS-mediated transformation of mouse embryonic fibroblasts (Shvarts et al. 2002). Using peptide inhibitors and small molecules that interfere with the ability of BCL6 to recruit essential corepressors, we and others recently demonstrated that therapeutic targeting of BCL6 in BCL6-dependent malignancies is feasible. Here we tested the hypothesis that BCL6 represents a therapeutic target in multiple subsets of ALL, including ALL with RAS pathway lesions.Results: In support of this hypothesis, we found that inducible expression of oncogenic NRASG12D increased BCL6 mRNA levels by ~350-fold (qRT-PCR) and protein levels by ~50-fold (Western blot). Upregulation of BCL6 in response to NRASG12D activation was sensitive to treatment with the MEK kinase inhibitor PD325901 and correlated with levels of phospho-ERK downstream of MEK. These findings suggest that BCL6 expression is a result of ERK activation downstream of oncogenic NRASG12D and MEK in ALL cells. To verify this observation in patient-derived cells, we compared ALL cells that were isolated at the time of initial diagnosis (D), and at the time of relapse (R) from the same patient. Interestingly, the patient had acquired a KRASG12V mutation at the time of relapse. As a likely consequence, we found both hyper-phosphorylation of ERK and overexpression of BCL6 in the relapse cells (KRASG12V), but not in diagnosis sample (KRAS wild-type). R-ALL cells harboring the KRASG12V mutation were more sensitive to the treatment with the MEK inhibitor PD325901 and the BCL6 peptide inhibitor RI-BPI than D-ALL cells. BCL6 inhibition also markedly increased survival rate of NOD/SCID mice xenografted with R-ALL cells. These results suggested BCL6 served as an important contributor to RAS -mediated transformation.To further study the mechanistic role of BCL6 in RAS-mediated pre-B cell transformation, we tested its function in a mouse ALL model. Pre-B cells from both Bcl6+/+ and Bcl6-/- mice could be transduced by NRASG12D and achieved growth-factor independence under cell culture conditions. However, Bcl6-/- NRASG12D ALL cells failed to initiate fatal leukemia in NOD/SCID transplant recipient mice, whereas Bcl6+/+ NRASG12D ALL cells gave rise to lethal leukemia in all transplant recipients. Studying Cre-mediated deletion of Bcl6-fl/fl alleles in a complementary mouse model revealed that continuous presence of Bcl6 function is required for normal proliferation of ALL cells. Cre-mediated ablation of BCL6 in NRASG12D driven ALL induced rapid cell death and completely abrogated the ability of NRASG12D ALL cells to form colonies.Conclusion: These results support that BCL6 is not only required for the initiation of RAS-transformed ALL in vivo but also for the maintenance of fully established RAS-driven leukemia. The findings provide genetic evidence for BCL6 function as a critical cofactor of RAS-mediated transformation in human ALL. Inhibition of BCL6 in RAS-driven ALL may be useful to prevent leukemia relapse after initial remission (Bcl6-dependent leukemia-initiation) and also to achieve remission by combining conventional cytotoxic therapies with currently available BCL6 inhibitors (e.g. RI-BPI peptide inhibitor or FX-1085 small molecule inhibitor). DisclosuresNo relevant conflicts of interest to declare.

Immunotoxin Activity Against B-Lineage Leukemia and Lymphoma Is Exposure Time Dependent: Implications for Trial Design

Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT) Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (HCL) (Kreitman et al 2012), but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR 2014). In contrast, HA22 is active vs primary blasts from 24/35 (68%) children with ALL in vitro (Mussai et al 2010). The apparent discrepancy between clinical response rates and in vitro cytotoxicity against ALL might be explained by the short serum half-life observed in clinical trials (approximately 1 hour). We hypothesized that blasts need to be exposed to RIT for longer in order to die. We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11, which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These modifications have been associated with pre-clinical improvements including reduced side effects, prolonged half-life, and diminished immunogenicity. LMB-11 achieved sustained CR in a Burkitt lymphoma animal model (Bera et al 2014). In the current study, we used LMB-11 and HA22 to evaluate activity against ALL and to try to identify methods to maximize in vitro cytotoxicity with the goal to increase clinical response rates in future clinical trials.Methods: Cytotoxicity was assessed with growth inhibition assays (WST-8). ALL cells were incubated with RIT for various times, washed, re-plated for a total of 72 h, stained for apoptosis by Annexin V-PE/7-AAD, and cell death analyzed by flow cytometry. NSG mice were inoculated with 5 million ALL cells IV on day 1 and treated with RIT from day 8 either IV or by continuous infusion with ALZET osmotic pumps implanted into the peritoneal cavity. Bone marrow (BM) samples were analyzed by flow cytometry for ALL infiltration and apoptosis.Results:In vitro cytotoxicity (IC50) of cell lines varied 210-fold (0.02 to 4.6 ng/ml). The time that cells had to be exposed to RIT to kill >90% of the cells varied greatly from <30 minutes to >4 days. We observed similar exposure time variability on 8 primary ALL samples. Despite this variability, for 57% (8/14) of all samples tested, the two toxins HA22 and LMB-11 were similar in the time to induce cell death. For 38% (5/14) of samples, HA22 was up to 10-fold more active and killed more rapidly than LMB-11. One example was KOPN-8, which was killed after less than 30 minutes exposure to HA22, but needed more than 9 hours exposure to LMB-11 for similar effects. The difference in exposure time dependent killing between the two toxins was related to intracellular RIT-processing. Using two newly developed ALL xenograft models with KOPN-8 or HAL-01, we found that response in vivo correlated directly with the time needed to kill the cells in vitro. Because RITs have a short serum half-life in mice (<30 minutes) but killing of KOPN-8 or HAL-01 cells needed 9 h or 24 h exposure to LMB-11, we replaced bolus dosing with small doses given at frequent intervals or with continuous infusion. The change in dosing strongly improved response of both KOPN-8 and HAL-01 xenografts (table 1).Conclusion: The previously unrecognized high variability of the time a CD22-targeting immunotoxin requires to kill ALL blasts provides a potential explanation for differences between in vitro cytotoxicity and the response rates observed in clinical trials. Our data indicate that ALL patients might show improved responses to anti-CD22 immunotoxins if treated with continuous infusion rather than the currently used bolus injection.Table 1In vivo response to immunotoxin improved with change in dosing.ALL Xenograft Cell lineRITChange in dosingChange in responseKOPN-8LMB-11QOD → frequent injectionsSD → CRHAL-01HA22QOD → continuous infusionPD → PRRIT = recombinant immunotoxin, QOD = every other day, IP = intraperitoneal, SD = stable disease, CR = complete response, PD = progressive disease, PR = partial response. DisclosuresWayne:Medimmune: Honoraria, Other: travel support, Research Funding; NIH: Patents & Royalties; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding. Pastan:NIH: Patents & Royalties: Coinventor on NIH Patent.

Increased Frequency of Cells with Activated Ribosomal Protein S6 at Diagnosis Associates with MRD Positivity and Relapse in Childhood BCP ALL

Numerous studies have demonstrated the association between minimal residual disease (MRD) positivity and risk of eventual relapse for children with B cell precursor acute lymphoblastic leukemia (BCP ALL). Yet despite varied methods to detect MRD (flow cytometry, PCR, sequencing), we still lack knowledge of the biology of drug resistant cells that mediate relapse.Performing single cell proteomic analysis using mass cytometry in a clinically annotated cohort of diagnostic samples, we have identified a phosphorylated ribosomal protein S6 (prpS6) signaling phenotype associated with MRD positivity and relapse. This phenotype has been observed in matched diagnostic and MRD samples and notably, in matched diagnosis/relapse pairs, we have observed an enrichment for this population at relapse.Methods Diagnostic BCP ALL samples from patients treated on the AIEOP protocols or healthy bone marrow were assayed either in basal condition or after ex vivo treatment with tyrosine kinase inhibitors (Dasatinib, BEZ-235). Samples were analyzed using mass cytometry for expression of 40 functional and/or phenotyping proteins. Mass cytometry data was processed and analyzed using Cytobank. Statistical analysis was performed using GraphPad Prism software. Significance was determined using student's t-test or one way ANOVA.Results Of the nine functional proteins measured in this study, rpS6 demonstrated, in resting cells from patients, the most variable levels of phosphorylation. Across the cohort the frequency of cells with activated rpS6 in the basal state varied between 4%-86% (median 24.7%), depending on the patient. This level was significantly higher than in healthy bone marrow controls (median 10.7%; p=0.0009). In BCP ALL samples the cells with activated rpS6 expressed higher levels of CD19 (p=0.0001), IgH (p=0.0059), and PAX5 (p=0.0021) when compared to cells without activated rpS6, suggesting a more mature phenotype for the prpS6+ blasts. These cells also had significant activation of pCREB (p<0.0001). Treatment with BEZ-235, a PI3K/TORk inhibitor, or Dasatinib, dual ABL/SRC kinase inhibitor, was able to significantly decrease the frequency of cells with activated prpS6 by inhibiting the phosphorylation of rpS6 after 30-minute ex vivo treatment.Patients with intermediate or high risk MRD status (per clinical protocol definitions) had an increased frequency of cells with activated rpS6 at diagnosis compared with patients with standard risk MRD (30% vs 13.9%; p=0.02). For patients for whom long term follow up data was available (n=31), patients that went on to relapse (n=9) compared to those in remission (n=22) had a statistically significant increased frequency of cells with activated prpS6 (p=0.0391).To determine whether activation of prpS6 was important for cell survival, we identified two cells lines amongst 7 BCP ALL cell lines that contained this high basal prpS6 subpopulation: NALM6 and SUP-B15. Treatment of these cells with Dasatinib in combination with the BEZ-235 or the MEKi PD0325901 resulted in decreased viability (6%-25% in TKI treated vs. 84% in untreated) and decreased activation of prpS6 and pCREB at 72 hours. These results support the hypothesis that the prpS6 high phenotype is important for cell survival.Given the association of activated rpS6 at diagnosis with detectable MRD and relapse, we examined an additional cohort of 9 matched samples from diagnosis Day 8 and Day 15 post induction therapy. In these samples, it was noted that by Day 8, there was a significant enrichment for cells with activated rpS6 (p=0.0009). In paired diagnosis-relapse samples, in 5 of 6 pairs, there was an enrichment for cells with activated rpS6atrelapse, all suggesting that rpS6 is associated with cells that are drug resistant. Mean prpS6 was significantly higher at relapse compared to diagnosis (p=0.035). Further studies will be critical to determine whether the rpS6 signature is proximal to causal events driving resistance.Significance We describe biochemical features of a cellular population observed at diagnosis across genetic subtypesassociated with MRD positivity and relapse that is enriched in early MRD and relapse samples in BCP ALL. Ribosomal protein S6 is a target of PI3K, MEK/ERK signaling and the B cell receptor pathway. Inhibition of this signaling results in cell death in model cell lines and suggests a role for therapy directed at this target in BCP ALL. DisclosuresDavis:Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

Epigenetics Directing Therapy in Acute Lymphocytic Leukemia: Emerging Roles and New Therapies

In the last 15 years, our understanding of the contribution of epigenetic mechanisms to ALL leukemogenesis has increased exponentially. Epigenetic alterations are particularly interesting from a therapeutic standpoint, as they are potentially reversible and amenable to pharmacologic modulation. Indeed, inhibitors of DNA methylation, histone deactylation and histone methylation all have entered clinical trials, and early results for several of these agents are encouraging. Incorporating epigenetic modulation into ALL therapy may therefore hold great promise in improving tolerability and outcomes. Specifically, this session will address: - Patterns of aberrant DNA methylation and clinical trials combining "demethylating" agents with standard chemotherapy for relapsed ALL.DNA methylation patterns have been shown to correlate with outcome. Basic studies failed to conclusively link the regulation of specific genes or pathways to aberrant DNA methylation, and upfront methylation patterns poorly predict response to demethylating agents. Nevertheless, several clinical trials combining demethylating agents and chemotherapy in relapsed ALL are ongoing, with encouraging early results. These included responses of patients treated with decitabine and HyperCVAD who had previously failed to respond HyperCVAD alone. - Patterns of aberrant histone acetylation, the biology of histone acetyl transferases (HATs) and histone deacetylases (HDACS) in ALL, and clinical trials modulating histone acetylation. Histone acetylation patterns and a high frequency of deletions of the HAT CBP at relapse suggest that aberrant histone acetylation contributes to relapse and resistance. HDAC inhibitors show efficacy on ALL cells in preclinical studies, and clinical trials are ongoing. - The role of the histone acetylation "reader" protein Brd4 in ALL. In addition to "writers" (HAT) and "erasers" (HDACs), readers of histone acetylation may play a role in leukemia. Inhibition of Brd4 showed activity in several lymphoblastic leukemia models, and two Brd4 inhibitors are currently in clinical trials for relapsed/refractory hematologic malignancies including ALL. - Patterns of aberrant histone methylation, biology and therapeutic possibilities. Aberrant histone methylation is also emerging as a contributor to leukemogenesis, and may offer opportunities for therapeutic intervention. Loss of function mutations of the H3K27 histone methyltransferase EZH2 or other members of its complex (SUZ12, EED) are often found in early T-cell precursor ALL (ETP), and loss of EED or EZH2 contributes to aberrant T-cell development and leukemic transformation in mouse models. Gain of function of the H3K36 histone methyltransferase NSD2 have recently been described in pre-B ALL, raising the obvious question whether these leukemias depend on H3K36 hypermethylation, and NSD2 inhibition would have a therapeutic effect. Finally, aberrant recruitment of the histone methyltransferase DOT1L is observed in MLL-rearranged ALL, and an inhibitor of DOT1L is in clinical trials for MLL-rearranged malignancies, including ALL. Basic and translational studies support a critical role for epigenetic mechanisms in ALL leukemogenesis, drug resistance and relapse. Early results from clinical trials demonstrate that pharmacologic modulation of epigenetic modifiers can produce clinically meaningful responses. The next few years will likely see an increased number of compounds that modulate epigenetics enter clinical trials. Current assignment of patients to studies/compounds is crude, based on tumor type and status (i.e. "relapsed hematologic malignancy") or cytogenetics (i.e. MLL-rearrangement), rather than epigenetic profiling or an understanding of the biology that drives an individual patient's leukemia. As our diagnostic and therapeutic tools improve, epigenetic modulation may become an important component of ALL therapy. Disclosures Off Label Use: This presentation will include discussion of the use of decitabine, azacitidine, vorinostat, rhomidepsin and EPZ-5676 for ALL..

T-Cell Acute Lymphoblastic Leukemia Patients with Mutations in IL7Ra or Downstream RAS-MEK or PI3K-AKT Can be Collectively Targeted By Combination of RAS and AKT Inhibitors

Background:Pediatric T-cell acute lymphoblastic leukemia patients frequently harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways.Aims:We aimed to determine the overall incidence of mutations in IL7Ra and downstream signaling components in a large cohort of pediatric T-ALL patients. In order to find better treatment options for patients with these mutations, we analyzed the effect of selected IL7Ra-pathway inhibitors-individually and in combinations-on downstream signaling and cytotoxicity in Ba/F3 cells expressing each of the mutations.Methods:We sequenced 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1, JAK2, JAK3, TYK2) and hotspot regions of N-RAS and K-RAS. We adapted the IL3-dependent Ba/F3 cell line to express mutant or wild type genes upon induction by doxycycline and assessed cell viability and signaling in the absence of IL3. Various IL7Ra-pathway inhibitors were tested using this system, and the synergy of combined inhibitors was determined by comparing the dose-response curve of different ratios of IC50-based inhibitor concentrations to the curves for each of the single inhibitors. The Combination Index was calculated using Calcusyn™ software.Results:IL7Ra, JAK, RAS, AKT and PTEN mutations are present in approximately 45% of patients and occur in a predominantly mutually exclusive fashion, suggesting they share aberrant activation of similar downstream targets. We found JAK1, JAK3 and RAS mutations as previously reported, but also identified new JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. A novel three-dimensional model of JAK1 reveals that mutations in JAK molecules affect important amino acids that are involved in the interaction between the pseudokinase and kinase domains, facilitating constitutive kinase activity. In our doxycycline-inducible IL3-dependent Ba/F3 system, expression of mutant genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth through activation of the RAS-MEK-ERK and PI3K-AKT pathways. We used this system to test the sensitivity to pharmacological inhibitors; IL7Ra and JAK mutant Ba/F3 cells are sensitive to JAK inhibition, so JAK inhibitors such as ruxolitinib may offer therapeutic potential for IL7Ra, JAK1 or most JAK3 mutated T-ALL patients. The RAS and AKT mutants respond to RAS-MEK and PI3K-AKT-mTOR inhibition, respectively, but are-as expected-insensitive to JAK inhibition. Remarkably, IL7Ra and JAK mutants are relatively resistant to downstream RAS-MEK-ERK or PI3K-AKT-mTOR inhibition, indicating that inhibiting just one of these downstream pathways is insufficient. We provide evidence of (cross-)activation of the alternate pathway when one of these pathways is inhibited. Combined inhibition of MEK and PI3K/AKT synergistically prevents proliferation of the IL7Ra- and JAK-mutants by efficiently blocking both downstream signaling pathways. Furthermore, this combined inhibition is cytotoxic in two out of five tested primary T-ALL specimens.Summary/Conclusion:We show that the combined inhibition of MEK and PI3K/AKT leads to strong and synergistic cytotoxic effects in the IL7Ra and JAK mutants and efficiently blocks signaling downstream of both pathways. This inhibitor combination is effective in two out of five primary T-ALL samples. Therefore, the cytotoxic effects of synergistic MEK and PI3K/AKT inhibition should be further explored as a therapeutic option for (relapsed) ALL patients. DisclosuresBuijsman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

Nuclear Export Inhibitor KPT-8602 Is Highly Active Against Leukemic Blasts and Leukemia-Initiating Cells in Patient-Derived Xenograft Models of AML

Currently available combination chemotherapy for AML induces complete remission in about 75% of younger and 45% of older patients, but often fails to induce long-term disease-free survival and is toxic to normal hematopoietic cells. The frequent disease relapse observed in chemotherapy treated AML patients is thought to occur through the inability of the existing drugs to specifically target the self-renewing leukemia-initiating cells (LICs). These cells have the functional capacity to replenish AML blasts. An attractive target for AML therapy is the nuclear export protein exportin 1 (XPO1), which is functionally required for continuous nuclear export of a subset of proteins and mRNAs. Small molecule Selective Inhibitor of Nuclear Export (SINE) compounds covalently bind to Cysteine528 in the cargo-binding groove of XPO1 and inhibit nuclear export. The orally bioavailable clinical SINE compound, selinexor (KPT-330), is currently in Phase 1 and 2 clinical trials in adult patients with AML (NCT01607892, NCT02088541, NCT02249091, NCT02403310, NCT02093403, NCT02485535, NCT02299518, NCT02416908, NCT02212561, and NCT01607892) and in a Phase 1 trial for relapsed childhood ALL and AML (NCT02091245). The results of these trials are encouraging, as they have demonstrated that selinexor alone or in combination is active in inducing remission in patients with relapsed or refractory AML. Here, we explore the anti-leukemia activity of a next generation SINE compound, KPT-8602.In preclinical toxicology studies as well as clinical trials, noted adverse events for selinexor are anorexia with weight loss, fatigue and thrombocytopenia, which limit the frequency of dosing to no more than every other day, three times a week. KPT-8602 is orally bioavailable and has similar pharmacokinetic properties to selinexor, with reduced brain penetration. Preliminary toxicology studies in rats and monkeys suggest that KPT-8602 has a substantially better tolerability profile, with reduced fatigue, thrombocytopenia, and anorexia compared to selinexor. Thus, KPT-8602 can be dosed orally on a daily basis for 5 days each week. The increased drug exposure with daily dosing and better tolerability of KPT-8602 leads directly to superior efficacy in pre-clinical models compared with selinexor.To define the anti-leukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant pre-clinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts were treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 was highly active against blast cells from three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML)), as evidenced by a reduction in leukemic engraftment in primary mice after treatment (Fig. 1A-C). At these doses, KPT-8602 demonstrated higher anti-leukemic activity than selinexor, with no leukemic cells detected in the bone marrow of two of the eight AML-CN PDX treated mice (Fig. 1A-C). In secondary transplantation assays, KPT-8602 greatly reduced the frequency of LICs in the PDX model derived from AML-CN cells, indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs (Fig. 1D). In mice engrafted with normal cord blood human CD34+ cells, KPT-8602 reduced bone marrow human CD45+ cell numbers (Fig. 1E), but the impact on normal hematopoietic stem and progenitor cells, as determined by secondary transplantation assays (Fig. 1E), was modest when compared to the LIC reduction after KPT-8602 treatment. These findings demonstrate that KPT-8602 has better tolerability, perhaps through decreased CNS penetration, and is better at eliminating both AML LIC and blast cells when compared to selinexor. Therefore, KPT-8602 should be tested for efficacy and tolerability in patients with relapsed and refractory AML, with the goal to overcome a difficult obstacle in curing AML; destroying the LIC compartment while sparing normal hematopoietic cells. [Display omitted] DisclosuresEtchin:Karyopharm: Research Funding. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Ellis:Karyopharm Therapeutics Inc: Employment. McCauley:Karyopharm Therapeutics, Inc: Employment. Stone:Agios: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Karyopharm: Consultancy; Celator: Consultancy; Novartis: Research Funding; Merck: Consultancy; Juno: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Roche/Genetech: Consultancy; Celgene: Consultancy. DeAngelo:Bristol Myers Squibb: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Ariad: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Look:Karyopharm Therapeutics, Inc: Research Funding.

Abstract A157: Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia

Abstract Objective: The Pseudomonas exotoxin (PE)-based anti-CD22 recombinant immunotoxin (RIT) Moxetumomab pasudotox (HA22) has a response rate of 86% in hairy cell leukemia (Kreitman et al 2012), but only about 30% of children with multiply relapsed ALL respond to HA22 treatment (Wayne et al AACR 2014). We attempted to improve activity against B-cell malignancies with a modified immunotoxin, LMB-11, which is missing domain II, has an Fab instead of a dsFv, and has 7 mutations in domain III. These modifications have been associated with reduced side effects, prolonged half-life, diminished immunogenicity, and better responses in in a Burkitt lymphoma animal model (Bera et al 2014). Activity of HA22 and LMB-11 on ALL cells varies significantly in vitro and in vivo (Müller et al AACR 2015). With the goal to improve activity against ALL, we combined CD22-targeting RITs with the multi-kinase inhibitor H89. H89 inhibits PKA (135 nM) and other kinases such as S6K1 (80 nM), MSK1 (120 nM), and ROCK2 (270 nM) (Davies et al 2000). Methods: In vitro cytotoxicity (IC50) was determined by WST-8 assay, and cell death by Annexin V-PE/7-AAD flow cytometry. An in vitro screen for H89 targets was performed by Reaction Biology Corp. Results: 10 uM H89 specifically enhanced activity of LMB-11 or HA22 10- fold on KOPN-8; activity of controls were unchanged. H89 also increased RIT activity by 5 to 10-fold on the ALL cell lines REH, SEM, HAL-01, and Nalm-6 and on 4 primary ALL samples. H89 did not change the rates of RIT-internalization or of furin-cleavage in KOPN-8. However, the time to ADP-ribosylate EF2 and arrest protein synthesis was shortened from 10 to 4 h with H89. We tested phosphorylation states of downstream targets in H89 treated KOPN-8 cells. Phosphorylation level of the PKA targets p-CREB or p-ATF2 were not reduced by H89. A high baseline phosphorylation of rpS6 or GSK-3β at the S6K1 target site was decreased to undetectable levels 15 minutes after addition of H89 to KOPN-8 and MCL1 fell by 50% within 15 minutes. In KOPN-8, a low dose of 5 ng/ml LMB-11 or 10 uM H89, respectively lowered MCL1 but did not induce apoptosis; MCL1 was undetectable and PARP-cleavage induced when H89 and LMB-11 were combined. The more specific S6K1 inhibitor PF-4708671 showed only 2-fold enhancement of RIT-induced cytotoxicity. The ROCK2 inhibitor Stemolecule similarly enhanced cytotoxicity 2-fold. MSK-1 inhibition or PKA-inhibition as well as PKA activation did not alter RIT activity. We performed a kinase screen on 359 kinases of which 21 were inhibited more than 90% by H89. None of the additionally identified kinases showed enhancement of RIT activity once blocked with relevant inhibitors. Despite combinations of small molecule inhibitors (e.g. ROCK2 and S6K1; GSK-3β and ROCK2), we were not able to mimic the 10-fold enhancement of RIT-induced cytotoxicity observed with H89. Conclusion: H89 strongly enhances CD22-targeting RITs against ALL. The increase in activity was in part due to inhibition of S6K1 and ROCK2 and was reflected by a faster onset of ADP-ribosylation of EF2 indicating improved trafficking of RITs through the various intracellular compartments. H89 and RIT lowered MCL1 levels by a distinct mechanism and synergistically induced intrinsic apoptosis. Further pre-clinical testing to assess in vivo efficacy of this combination is ongoing. Citation Format: Fabian Mueller, Xiu Fen Liu, Alan S. Wayne, Ira Pastan. Multi-kinase inhibitor H89 enhances activity of pseudomonas exotoxin-based immunotoxins targeting acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A157.

PCN78 - Retrospective Chart Review of Hospitalisations During Chemotherapy for Adult Patients with Ph-Negative B-Precursor Relapsed or Refractory (R/R) Acute Lymphoblastic Leukaemia (All) In Italy

– 18 years of age or older – hospitalised for management of at least one episode of R/R ALL; diagnosis of Ph-negative B-precursor ALL – relapsed with first remission lasting less than 12 months – relapsed after first salvage therapy – relapsed any time after haematopoietic stem cell transplant HSCT – or refractory to primary induction or salvage therapy – electronic medical record or chart available for review and data collection – not enrolled in either blinatumomabor inotuzumab ozogamicin-related clinical trials during the study period • The study period was from 2003 to 2014. Patients with R/R ALL were screened from October 2013 going backwards until at least 30 eligible patients were identified. • Patient data were collected from the index date until the patient died or was lost to follow-up, and outcomes were evaluated during pre-specified time periods (Figure 1). – The index date was the first time the ALL patient was recorded as refractory or relapsed, according to the inclusion criteria. – The chemotherapy period was pre-specified to represent the time during which the most intensive chemotherapy is assumed to be administered but before transplant. The chemotherapy period was defined as the first chemotherapy date after the index date to the earliest of either death, loss to follow-up, last chemotherapy dose plus 30 days, or initiation of HSCT. Since the ultimate goal of therapy is to send patient to HSCT, initiation of HSCT was chosen as the end of the intensive chemotherapy period. – For patients who received HSCT after the index date, the HSCT period was defined as the time from starting HSCT to the earliest of death, loss to follow-up, or relapse of ALL. METHODS • To quantify hospitalisations and reimbursements among adults with PhR/R B-precursor ALL treated with current salvage chemotherapies in Italy.

Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

The Evolutionary Trajectory from Diagnosis to Relapse in Pediatric ALL

The Evolutionary Trajectory from Diagnosis to Relapse in Pediatric ALL

Abstract 3277: Optimized treatment schedule with the new generation CD22-targeting immunotoxin LMB11 induces near MRD-negativity in an ALL mouse model

Abstract Objective: The Pseudomonas exotoxin (PE) based anti-CD22 immunotoxin Moxetumomab pasudotox (HA22) has a response rate of 95% in hairy cell leukemia (HCL) (Kreitman et al 2012), but only about 25% of children with multiply relapsed ALL achieve a complete response (CR) (Wayne et al AACR 2014). HA22 is highly cytotoxic to B-lineage leukemia and lymphoma lines in vitro and in subcutaneous mouse models (Onda et al 2011). HA22 is also active vs primary blasts from 24/35 (68%) children with ALL (Mussai et al 2010). Since there is a significant difference between ALL patient cells in vitro and response rate in patients, we have investigated possible mechanisms of HA22 resistance in mice to aid development of methods to increase response rates in humans. Methods: We used a modified anti-CD22 immunotoxin, LMB11, which can be safely given to mice at a 10-fold higher dose than HA22 (Bera et al Leuk Lymph 2014). NSG mice were inoculated with 5 million KOPN8 cells I.V. on day 1 and treated on days 8, 10, 12, 14 and 16 with 50 μg LMB11. Bone marrow (BM) samples were analyzed by FLOW for KOPN8 infiltration and apoptosis. Bio-distribution studies used Alexa647-coupled LMB11 and Alexa647 beads to quantify number of LMB11 molecules taken up per cell. Cells were treated with LMB11 for various times, washed, re-plated for 3d, and cell death measured by Annexin V-PE/7-AAD in Flow. Results: KOPN8 cells are very sensitive to LMB11 (IC50, 0.5 ng/ml) in vitro. Maximal ADP-ribosylation of EF2 occurs at 6h, protein synthesis stops at 8h, MCL1 levels fall at 8h, and PARP cleavage begins at 10h. Untreated KOPN8-inoculated NSG mice survived 21.5 d. When treated with 50ug x 5 of LMB11, mice survived 30 d. There was no significant apoptosis of KOPN8 cells in murine BM during LMB11 dosing. ALL BM infiltration remained at 5% during dosing. Four days after the last dose, BM infiltration increased rapidly. Cellular uptake of LMB11 was 200-fold over the amount required to kill cells in vitro. To identify potential resistance mechanisms, we treated KOPN8 for various times in vitro and found that KOPN8 needed a 9h exposure to LMB11 for &amp;gt;95% of cells to die. There was p38 MAPK activation at 6h, which is probably responsible for cell cycle arrest of KOPN8 cells in the BM. Because LMB11 has a short half-life (30 min) in mice and because long exposure time is needed to induce apoptosis in vitro, we treated with 4 doses of 25 ug LMB11 every 3h for 3 days to maintain adequate blood levels. This resulted in near MRD-negative BM (0.004%) by flow. We screened many drugs for their ability to shorten the exposure time needed to induce &amp;gt;95% cell death by LMB11 and found that H89, nilotinib, imatinib and verapamil were active. Conclusion: KOPN8 cells need prolonged immunotoxin exposure to drive cells into apoptosis. HA22 given more frequently or combined with sensitizing agents might increase CR rates in patients with ALL. Citation Format: Fabian Mueller, XiuFen Liu, Alan Wayne, Ira Pastan. Optimized treatment schedule with the new generation CD22-targeting immunotoxin LMB11 induces near MRD-negativity in an ALL mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3277. doi:10.1158/1538-7445.AM2015-3277

Abstract 4445: Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells

Abstract Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are toxic to normal tissues, prompting the need to develop new targeted therapies. The frequent disease relapse that is observed in patients with AML is thought to occur because of the inability of the existing drugs to target the self-renewing leukemia-initiating cells (LICs). An attractive new strategy for AML therapy is inhibition of the nuclear export protein exporter 1 (XPO1), or CRM1. XPO1 regulates export of proteins that contain leucine-rich nuclear export signals (NES), including protein adaptors that mediate transport of RNA. XPO1 cargo encompass tumor suppressor proteins, cell cycle regulators, and apoptotic proteins. Recently, small molecule inhibitors of nuclear export (SINE) that inhibit the export function of XPO1 by targeting Cys528 in its NES-binding groove, were developed using an in silico molecular modeling. Selinexor (KPT-330), the orally bioavailable SINE compound, is in Phase 1 and 2 studies in adult patients with AML (NCT01607892 and NCT02088541) and in a Phase 1 study for relapsed childhood ALL and AML initiated in March 2014 (NCT02091245). To define the anti-leukemic activity of selinexor against primary AML blasts and LICs in a clinically relevant setting, we established mouse models of primary human leukemia, or patient-derived xenografts (PDX), in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcγnull (NSG) mice. Mice engrafted with leukemic blasts were treated with either vehicle or selinexor. Selinexor was highly active against blast cells from two of the three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN) and complex karyotype AML (AML-CK1 and AML-CK2)), as evidenced by a reduction in leukemic engraftment in primary mice after 4 weeks of treatment. Secondary transplantation assays indicated that selinexor greatly reduced the frequency of LICs in PDX models derived from all three patients (6- to 430- fold reduction compared to controls), indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs. These findings show that selinexor has potent activity against LICs, even when it has only moderate activity against the bulk AML cell population. Furthermore, preliminary results of combination studies of selinexor with Ara-C, a standard chemotherapeutic agent, demonstrate synergistic effect of the two drugs against LICs in a PDX model of AML-CN. Importantly, 4 weeks of selinexor treatment demonstrated minimal toxicity in mice engrafted with normal human CD34+ hematopoietic cells. These findings demonstrate that inhibition of nuclear export with selinexor overcomes an important obstacle to cure of AML, which is to destroy the very critical LIC compartment while sparing normal hematopoietic cells. Citation Format: Julia Etchin, Bonnie Thi Le, Alla Berezovskaya, Amy S. Conway, Weihsu C. Chen, Alex Kentsis, Marc R. Mansour, Richard M. Stone, Ilene A. Galinsky, Daniel J. DeAngelo, Dilara McCauley, Michael Kauffman, Sharon Shacham, Jean CY Wang, Andrew L. Kung, Thomas Look. Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2015-4445

Research Progress on Expression Regulation, Function and Clinical Significance of CASP8AP2 Gene

We systematically reviewed the results of the studies on expression regulation, biological functions, and clinical prognostic significance of CASP8AP2 gene. At present, the studies showed that the expression of CASP8AP2 gene was regulated by Homeobox proteins and DNA methylation, and could be silenced by miRNA-210. This protein was involved in apoptosis mediated by FAS and TNFα, NF-κB activation mediated by TNFα, regulation of gene expression induced by glucocorticoid and mineralocorticoid receptor, comprising Cajal body and histone locus body, transcription of replication-dependent histone, 3' end processing of histone, regulation of S phase progression, in addition to functioning as coactivator of transcription factors c-Myb and p73 to activating many genes' expression. On the other hand, low expression of CASP8AP2 gene was associated with relapse in childhood ALL. The deletion of this gene was related to the poor prognosis of children with T-ALL and T lymphoblastic lymphoma. Furthermore, 3 SNPs in this gene were possibly correlated with genesis of diffuse large B cell lymphoma and childhood leukemia. In conclusions, CASP8AP2 was a multifunctional protein. It could function to regulate cell proliferation, apoptosis, and gene expression. In childhood hematological malignancies, CASP8AP2 was a promising molecular marker with prognostic significance. Some SNPs were possibly correlated with leukemo- and lymphomogenesis.

Presence of CD34(+)CD38(-)CD58(-) leukemia-propagating cells at diagnosis identifies patients at high risk of relapse with Ph chromosome-positive ALL after allo-hematopoietic SCT.

Relapse of Ph chromosome-positive ALL (Ph(+)ALL) results from the persistence of leukemia-propagating cells (LPCs). In Ph(+)ALL, a xenograft assay recently determined that LPCs are enriched in the CD34(+)CD38(-)CD58(-) fraction. Therefore, the prognostic significance of LPCs in Ph(+)ALL subjects after allogeneic hematopoietic SCT (allo-HSCT) was investigated. A total of 80 consecutive adults with Ph(+)ALL who underwent allo-HSCT were eligible. A multi-parameter flow cytometry analysis examining CD58-FITC/CD10-PE/ CD19-APC-Cy7/CD34-PerCP/CD45-Vioblue/ CD38-APC on gated leukemia BM blasts was performed at diagnosis. Based on the original blast phenotypes, subjects were stratified into the CD34(+)CD38(-)CD58(-)group (N=15) and other phenotype group (N=65). During minimal residual disease monitoring, significantly higher levels of BCR/ABL transcripts were detected in subjects in the CD34(+)CD38(-)CD58(-) group than in other phenotype group, especially at 3 months post HSCT. In addition, CD34(+)CD38(-)CD58(-)LPCs are directly correlated with a higher 3-year cumulative incidence of relapse (CIR) and worse leukemia-free survival (LFS) and OS. Multivariate analyses indicated that presence of CD34(+)CD38(-)CD58(-) LPCs at diagnosis, and BCR-ABL reduction at 3 months post HSCT were independent risk factors for relapse, LFS and OS. Our data suggest that presence of CD34(+)CD38(-)CD58(-) LPCs at diagnosis allows rapid identification of high-risk patients for relapse after allo-HSCT.

Results of a Feasibility and Phase II Study on Bortezomib (BTZ) in Pediatric Multiply Relapsed or Refractory Acute Lymphoblastic Leukemia: Complete Hematological Responses with a Modestly Intensive Regimen Including BTZ

▪Prognosis of refractory and relapsed ALL is poor and its improvement requires the introduction of novel agents with a new mechanism of action. Bortezomib (BTZ) as proteasome inhibitor is such an agent, and has been shown to be safe as single agent in phase I studies in children with either solid tumors (Blaney 2004) or leukemias (Horton 2007). Recently, Messinger (2012) reported in a single-arm study that BTZ can be combined safely with vincristine (VCR), dexamethasone (DXM), pegylated asparaginase (PEG-ASP) and doxorubicin and that the combination was remarkably effective in B-cell precursor ALL. BTZ has been reported to sensitise malignant cells to other agents, both in vitro for leukemias as well as for multiple myeloma patients in vivo. In patients with ALL, the sensitizing effect of BTZ regarding the efficacy of glucocorticoids (GC) has not been addressed yet. Considering the lack of any clinical experience with BTZ in children, we developed a European multicenter feasibility and phase II study in refractory or relapsed ALL, in which all patients receive BTZ, with day 8 peripheral blast count as primary endpoint. The study is performed in compliance with the Declaration of Helsinki. Patients are randomised for BTZ to be administered “early”, or “late”. Bortezomib is then given as iv push for 4 doses at 1.3 mg/m2/dose, thus in group “early” on days 1, 4, 8 and 11 and in group “late” on days 8, 11, 15 and 18. In addition, all patients receive DXM (10 mg/m2/day in 3 doses for 2 weeks, orally or iv) and VCR (1.5 mg/m2/dose with a maximum of 2 mg as 1-hour infusion on days 8 and 15), as well as one intrathecal administration of methotrexate (MTX, dose age-adjusted) on day 1. No ASP and no anthracycline is given. Cycles can be repeated in case of a good response. Eligible patients have 2nd or greater relapsed ALL, 1strelapsed ALL after allogeneic stem cell transplantation (allo-SCT) in CR1, or refractory first relapsed ALL, and bone marrow (BM) involvement and at least 100 leukemic cells per µl blood. Exclusion criteria include symptomatic CNS leukemia. It is planned to evaluate 24 fully evaluable randomised patients. This analysis, not being an official interim-analysis and not evaluating the primary endpoint of the study, focusses on haematological responses with this modestly intensive regimen. BM M1 indicates <5% blasts, M2 5-15% and M3>15% blasts.Between October 2010 and March 2014, a total of 21 eligible patients with information on response and/or toxicity after cycle 1 has been enrolled, 11 boys and 10 girls, median 9.8 years of age (range, 1.2 – 17.5). Most had second relapsed ALL (n=11), others first relapsed ALL following allo-SCT in CR1 (n=8) or refractory first relapsed ALL (n=2). Regarding efficacy, 17 patients were evaluable (1 patient no material received, 2 patients discontinued treatment due to toxicity, and 1 patient had early progression which led to treatment discontinuation). Day 22 BM showed M1 in 5, M2 in 6, and M3 in 6 patients. There was no relation between day 22 BM status and the administration of BTZ being early or late. There was no association between response and disease status, being either refractory or relapsed, although numbers are small. Two patients with a relapse after allo-SCT achieved BM M1, as well as 3 patients with a second or subsequent relapse. Four patients (23.5%) achieved hematological remission after cycle 1, while 11 out of 17 patients (65%) had a good initial response (day 22 BM M1 or M2). A total of 10 patients received a 2ndcycle and 5 out of them achieved a complete remission, 4 of whom already had a day 22 BM M1. Eight out of 21 (38%) patients suffered from grade 3 and/or 4 toxicity during cycle 1, and most frequently reported were pain (n=4), peripheral neuropathy and fatigue (n=2 each). After 2 cycles, grade 3/4 toxicity was reported in 3 out of 10 patients: one with peripheral neuropathy, one with peripheral neuropathy, haemorrhage and hematuria, and one with raised ALAT.Future analyses will focus on sensitisation to GC by BTZ and on the efficacy of BTZ in relation to PK and PD. Meanwhile, BTZ in combination with VCR, DXM and intrathecal MTX is effective in a significant subset of pediatric relapsed and refractory ALL, and repetitive cycles could be given in several children without undue toxicity.This research is financially supported by the Dutch Foundation Children Cancer-free (clinical research support) and by Janssen Pharmaceuticals (clinical research support and free drug). DisclosuresBaruchel:Janssen-Cilag: Consultancy.

Infectious Complications in Children with ALL Treated with ALL-IC-2009 Protocol: Multicenter National Study of Polish Society of Pediatric Hematology and Oncology

BACKGROUND. Infectious complications are a significant cause of morbidity and mortality in children receiving treatment for ALL. One of the major factors increasing the risk of bacteremia is the widespread use of central venous lines (CVL). In this study we hypothesized if there are any outcome predictors during bacteremia episodes during standard chemotherapy.OBJECTIVE. The aim of the study was to analyze epidemiology and impact of bacteremia as a main cause of death during induction and consolidation therapy of ALL in children. We assessed an influence of following factors: age, sex, time to infection, duration of antibiotic treatment, and relapse on survival in ALL children with bacteremia.METHODS. We reviewed 430 medical charts of children with ALL treated in Pediatric Hematological Centers in Poland according to ALL-IC-2009 protocol between January 2012 and December 2013. The group of 110 children (71 boys, 39 girls) with ALL with at least 1 episode of bacteremia was analyzed. The median age of the children was 7.5 years. Statistical analysis was performed using chi-squared and t-test when applicable to estimate the influence of different factors (age, sex, time to infection, period of treatment) for the outcome.RESULTS. The overall incidence of bacteremia among ALL children was 110/430 (25.6%). The most common cause of bacteremia (20%) was S.epidermidis (22%) and E.Coli ESBL(+). The median time to infection (TTI) was 4.9 months . There was no significant association between TTI and outcome of the patients (p=0.07), but there was a trend towards increased death rate during consolidation therapy. The median duration of infection treatment (MDT) was 13.6 days. MDT did not influence significantly death rate (p=0.31). Among all patients, 14 (12.7%) deaths were observed. The median age of children in that group was 9.1 years. In the analyzed period 19 cases of ALL relapse were observed in this study. In the relapse group there was significantly higher mortality rate than in the group with “de novo ALL” (p<0.01). Mortality rate was significantly higher among boys (p<0.01). There was no correlation between age of children and incidence of deaths (p=0.14).CONCLUSIONS. The incidence of bacteremia in children treated for ALL in Poland is 25.6%. Staphylococcus epidermidis and Escherichia Coli ESBL(+) are the mostcommon pathogens. Bacteria translocation from intestines to the blood might play a key role in development of blood-stream infection (BSI), thus the gut decontamination may be of high value in high-risk patients. Children with ALL relapse are at high risk of mortality due to bacteremia. Boys are more predisposed to life-threatening episodes and higher mortality due to BSI. Age is not a predictor factor for bacteremia related mortality. Children during consolidation chemotherapy of ALL require special attention to avoid bacteremia episodes. DisclosuresNo relevant conflicts of interest to declare.