Refractory Ph Research Articles

Venetoclax, Cladribine and Cytarabine for the Treatment of Relapse/Refractory Acute Lymphoblastic Leukemia: Interim Analysis of a Phase 2 Trial

Venetoclax, cladribine and cytarabine for the treatment of relapse/refractory acute lymphoblastic leukemia: interim analysis of a phase 2 trial Background: Philadelphia chromosome-negative acute B lymphoblastic leukemia (Ph- B-ALL) accounts for approximately 60-70% of adult B-ALL. Patients with relapse/refractory (R/R) Ph- B-ALL was reported to have a poor prognosis. Immunotherapies such as blinatumomab, CAR T-cell therapy achieved high response rate for Ph- B-ALL. However, salvage chemotherapies are also worth of investigation because many patients can't afford immunotherapies. We found that venetoclax, cladribine and low-dose cytarabine had synergistic cell toxicity against B-ALL cell line RS4;11 in our previously in vitro studies. Based on these findings, we designed a multicenter, phase 2 study utilizing the venetoclax, cladribine and cytarabine (CAV) regimen for the treatment of R/R B-ALL (NCT05657652). Methods: The CAV regimen consisted of cladribine 5 mg/m 2/day from day 1 to day 3 or day 5, cytarabine 20mg/m 2, q12h, for 7-10 days and venetoclax 100mg d1, 200 mg d2, 400 mg/day from day 3 to day 28. Bone aspiration evaluation was performed at 14-28 days after the completion of CAV regimen. Treatment responses were evaluated according to the NCCN guidelines. The threshold of MRD negativity was defined as less than 0.01% as detected by multiparameter flow cytometry. Results: Since February 2021, a total of 16 patients with R/R Ph- B-ALL were enrolled in this study. Baseline characteristics of these patients were summarized in Table 1. The median age was 36 years (range, 13-66 years), and 12/16 (75%) patients were male. Among them, 12 patients (75%) had relapsed Ph- B-ALL and 4 patients (25%) had refractory Ph- B-ALL. One patient had concurrent active central nervous system leukemia. One patient had a complex karyotype, and two patients had TP53 mutations. Treatment responses were shown in Table 2. 12/16 (75%) patients responded to CAV regimen after one cycle of CAV regimen, with 3/16 (18.75%) with complete remission (CR), 8/16 (50%) with CRi, and 1/16 (6.25%) with morphology leukemia-free state (MLFS). Among patients who achieved treatment response, 9/12 (75%) patients attained MRD negativity. During the treatment with the CAV regimen, one patient (1/16, 6.3%) developed grade 4 tumor lysis syndrome. 4/16 (25%) patients suffered grade 3 to 4 infections. Grade 1 to 2 gastrointestinal reactions and liver dysfunction were observed in 8/16 (50%) and 5/16 (31.3%) patients, respectively. The 60-day mortality rate was 6.3%. Two patients who relapsed after CAV treatment re-achieved remission after CART therapy. Nine patients received allo-HSCT after CAV regimen. With a median follow-up of 11 months (range, 0.3-28 months), the estimated 1-year overall survival rate was 76.2% and 1-year event-free survival rate was 38.4%. The survival swimming plot was shown in Figure 1. The median OS was not reached and the median EFS was 13 months. The cumulative relapse rate was 31.3% (5/16). At the last follow-up, two patients died of relapse, one patient died of cerebral hemorrhage and one patient died of sepsis. Conclusion: In summary, our results demonstrated that CAV regimen showed encouraging efficacy and safety in treating R/R Ph- B-ALL patients.

Unraveling the Mechanism of Action of Bispecific T-Cell Engagers in B-Cell Acute Lymphoblastic Leukemia Using Advanced Single-Cell Multiomics

Background Bispecific T-cell engagers (BiTEs) stimulate T lymphocytes proximate to tumor cells, inducing T-cell stimulation and tumor killing. Blinatumomab, an anti CD3-CD19, was the first to be approved with overall response rates of up to 80% in refractory B-cell Acute Lymphoblastic Leukemia (B-ALL). To overcome resistance and to develop next-generation BiTEs, we need to build on blinatumomab's success and better understand BiTEs mode of action and interaction with the tumor microenvironment (TME). Here, we apply advanced multiomics to examine blinatumomab's activation signatures and the sustained dynamics in the tumor and bone marrow (BM) immune TME. Methods Physically interacting Cells sequencing (PIC-seq) is an advanced multiomics technology deciphering the crosstalk between the cellular components of an immunological synapse (Cohen et al., 2022; Giladi et al., 2020). To assess Immediate activation signatures, we applied PIC-seq on a model of B-ALL cell lines (RS4;11, NALM-6) and lymphocytes obtained from healthy subjects, activated by blinatumomab, compared to no-treatment and activation with anti CD3-CD28 antibodies. To examine the Sustained effects on the immune TME, we performed a longitudinal single-cell RNA sequencing of the bone BM of adult B-ALL patients, before and after treatment with blinatumomab in the Division of Hematology in the Tel Aviv Sourasky Medical Center in 2021-22. Results Immediate activation Applying PIC-seq on n=16,543 CD3 and CD19+ cells, we identified the distinct gene expression of ‘blinatumomab-activated lymphocytes’ (Figure 1). Blinatumomab induced cellular synapses composed of specific T lymphocytes subsets along the activation continuum, namely: effector T cells (CCL5, GNLY, NKG7), early IFN-G activation (IRF1, GBP5), activated cytotoxic (Xcl1-2, IFN-G) and proliferating T cells (CD69, CCND2) . ‘Blinatumomab-activated lymphocytes’ were characterized by a unique activation signature: upregulation of cytotoxicity (Xcl1, IFN-G, GNLY), co-stimulation and B-T cells crosstalk (CD40, CD27, TNFRSF18), immune recruitment (CXCL10, CCL4) and antigen-presentation (TNFSF14, ITGB2) pathways. Late synapses also expressed immunoregulatory molecules (TNFAIP8L2). This signature differed from classical anti CD3-CD28 induced activation and is independent of the donor and the target-cell identity. The malignant cells' response varied: RS4;11 cells manifested cellular senescence, while NALM-6 upregulated INFG-induced apoptotic pathways, suggesting indirect killing in the mechanism of action of BiTEs. Data on the introduction of primary B-ALL patients' cells will be presented at the ASH. Sustained effects We profiled >11,000 cells from three patients: Patient 1, 31-year-old (y/o) female, primary refractory B-ALL (50% BM blasts), attaining transient MRD negativity. Patient 2, 71-y/o male, primary refractory Ph+ B-ALL (74% BM blasts) achieving reduction in MRD following first cycle. Patient 3, 71 y/o, Ph+ B-ALL, treated as consolidation therapy, achieving MRD negativity. Blinatumomab induced a dramatic perturbation in the lymphocytic and myeloid compartments of the BM and was highly patient-specific ( Figure 2). The BM lymphocytic compartment prior to initiation of blinatumomab in molecular remission (patient 3) was characterized by active T and Natural Killer cell states, compared to an abundancy of naïve lymphocytes in active disease (patients 1 and 2). There were no signs of T-cell exhaustion pre or post treatment in all patients. Post-treatment, CD8 T-Central Memory cells proliferated in all patients. Patients treated for active disease (patients 2 and 3), had an expansion of the Tregs population post-treatment. In the myeloid compartment, patients with active disease had more non-classical monocytes, known to correlate with poor prognosis. These were replaced by classical monocytes following treatment, in addition to the emergence of a macrophage-precursor population. Data on the BM and peripheral blood dynamics of an additional six patients will be presented at the ASH. Conclusions We present an advanced single cell dissection of blinatumomab-induced target-effector interaction, providing novel insights into the mechanism of action and TME dynamics to guide patient selection and next generation BiTEs. A larger patients' cohort and introduction of patients' cells to the PIC-seq in vitro model will be presented at the ASH.

Olverembatinib Treatment in Pediatric Patients With Relapsed Philadelphia-Chromosome-Positive Acute Lymphoblastic Leukemia

Treatment outcomes for children with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) remained poor despite the use of intensive chemotherapy, imatinib or dasatinib, and consolidative allogeneic hematopoietic cell transplantation. Oleverembatinib, a third-generation ABL inhibitor, was found to be highly effective and safe in adults with chronic myeloid leukemia and in some adults with relapsed or refractory Ph+ ALL. We reviewed the efficacy and safety profile of olverembatinib treatment in 6 children with relapsed Ph+ ALL and 1 with T-ALL and ABL class fusion, all of whom had previously received dasatinib or intolerance to dasatinib. The median duration of olverembatinib treatment was 70 days (range: 4-340) and the median cumulative dose was 600 mg (range: 80-3810). Complete remission with negative minimal residual level (<0.01%) was achieved in 4 of the 5 evaluable patients, 2 of whom were treated with olvermbatinib as a single agent. Safety profile in 6 evaluable patients was excellent with grade 2 extremity pain occurred in 2 patients and grade 2 myopathy of lower extremity and grade 3 fever in 1 patient each. Olverembatinib appeared to be safe and effective in children with relapsed Ph+ ALL.

RUNX1 transactivates BCR-ABL1 expression in Philadelphia chromosome positive acute lymphoblastic leukemia.

The emergence of tyrosine kinase inhibitors as part of a front‐line treatment has greatly improved the clinical outcome of the patients with Ph+ acute lymphoblastic leukemia (ALL). However, a portion of them still become refractory to the therapy mainly through acquiring mutations in the BCR‐ABL1 gene, necessitating a novel strategy to treat tyrosine kinase inhibitor (TKI)‐resistant Ph+ ALL cases. In this report, we show evidence that RUNX1 transcription factor stringently controls the expression of BCR‐ABL1, which can strategically be targeted by our novel RUNX inhibitor, Chb‐M'. Through a series of in vitro experiments, we identified that RUNX1 binds to the promoter of BCR and directly transactivates BCR‐ABL1 expression in Ph+ ALL cell lines. These cells showed significantly reduced expression of BCR‐ABL1 with suppressed proliferation upon RUNX1 knockdown. Moreover, treatment with Chb‐M' consistently downregulated the expression of BCR‐ABL1 in these cells and this drug was highly effective even in an imatinib‐resistant Ph+ ALL cell line. In good agreement with these findings, forced expression of BCR‐ABL1 in these cells conferred relative resistance to Chb‐M'. In addition, in vivo experiments with the Ph+ ALL patient‐derived xenograft cells showed similar results. In summary, targeting RUNX1 therapeutically in Ph+ ALL cells may lead to overcoming TKI resistance through the transcriptional regulation of BCR‐ABL1. Chb‐M' could be a novel drug for patients with TKI‐resistant refractory Ph+ ALL.

Inhibition of the RacGEF VAV3 by the small molecule IODVA1 impedes RAC signaling and overcomes resistance to tyrosine kinase inhibition in acute lymphoblastic leukemia

Aberrant RHO guanine nucleotide exchange factor (RhoGEF) activation is chief mechanism driving abnormal activation of their GTPase targets in transformation and tumorigenesis. Consequently, a small-molecule inhibitor of RhoGEF can make an anti-cancer drug. We used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like acute lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation–dependent RacGEF, as the target of the small molecule IODVA1. We show that through binding to VAV3, IODVA1 inhibits RAC activation and signaling and increases pro-apoptotic activity in BCR-ABL1-transformed cells. Consistent with this mechanism of action, cellular and animal models of BCR-ABL1-induced leukemia in Vav3-null background do not respond to IODVA1. By durably decreasing in vivo RAC signaling, IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal. Importantly, IODVA1 suppresses the leukemic burden in the treatment refractory pediatric Ph+ and TKI-resistant Ph+ B-ALL patient-derived xenograft models better than standard-of-care dasatinib or ponatinib and provides a more durable response after treatment withdrawal. Pediatric leukemia samples with diverse genetic lesions show high sensitivity to IODVA1 ex vivo and this sensitivity is VAV3 dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapy.

Inotuzumab Ozogamicin in Combination with Bosutinib for Patients with Relapsed or Refractory Ph+ ALL or CML in Lymphoid Blast Phase

Inotuzumab Ozogamicin in Combination with Bosutinib for Patients with Relapsed or Refractory Ph+ ALL or CML in Lymphoid Blast Phase

CML-089: Retrospective Independent Review of Arterial Occlusive Events (AOEs) in the Ponatinib Phase 2 PACE Trial (NCT01207440) in Patients with Philadelphia Chromosome Positive (Ph+) Leukemia

Objectives In the PACE trial 5-year analysis evaluating use of ponatinib in patients with refractory chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia, a 25% incidence of AOEs was identified (Cortes, Blood 2018) using Medical Dictionary for Regulatory Activities (MedDRA) preferred terms (PTs). We performed a retrospective review using a charter-based independent Adjudication Committee (AC) to better understand clinically relevant AOE rates in PACE. Methods The AC (3 cardiologists, 1 hematologist, and 1 neurologist) evaluated AOEs (identified using >500 terms) in PACE using established definitions developed by the American College of Cardiology/American Heart Association (ACC/AHA), US Food and Drug Administration (FDA), and Standardized Data Collection for Cardiovascular Trials Initiative (SCTI). The AC also reviewed patient profiles including event, severity, concomitant medication, and hospitalization data. These adjudicated results were compared with pre-adjudication MedDRA PT search results. The AC was blind to dose, dose modification, and investigator causality opinion. Events not associated with a cardiovascular etiology or that failed to meet the ACC/AHA, FDA, and SCTI definitions were determined by the AC not to be AOEs. Results This review included all 449 heavily pretreated patients with Ph+ leukemia enrolled in PACE (median age, 59 y; 47% female; 93% ≥2 tyrosine kinase inhibitors). At a median follow-up of 37.3 months, adjudicated AOEs occurred in 17.4% of the overall patient population compared with 24.7% pre-adjudication. The majority of the pre-adjudication serious AOEs were adjudicated as serious AOEs (20% pre-adjudication vs 16% adjudicated). The most common adjudicated AOE (>2%) was peripheral arterial occlusive disease. Exposure-adjusted incidence of newly occurring adjudicated AOEs decreased over time. Analyses by baseline risk factors demonstrated that patients with the following baseline risk factors had higher incidence rates of adjudicated AOEs than those without ischemic cardiac disease, hypertension, hypercholesterolemia, and diabetes. Conclusions Independent AC review of the PACE trial identified a lower rate of clinically relevant AOEs than previously reported, suggesting an earlier possible overestimation that may not accurately reflect the risk of AOEs with ponatinib. The ongoing ponatinib dose-ranging OPTIC study will further evaluate dose-dependent AOE incidence rates.

Huaier extract enhances the treatment efficacy of imatinib in Ik6+ Ph+ acute lymphoblastic leukemia

Philadelphia chromosome-positive (Ph+) is considered as a high risk of acute lymphoblastic leukemia (ALL). Tyrosine kinase inhibitors (TKIs) are tailored drug for Ph+ ALL, but Ik6 is associated with TKI resistance and poor outcome of Ph+ ALL. In the present study, we investigated the potential benefit of combination therapy with imatinib and Huaier extract, a traditional Chinese medicine, in Ik6+ Ph+ ALL. The Ik6+ Ph+ -ALL cell lines Sup-B15 or BV173 were treated with Huaier extract, imatinib or the combination of the two. Analysis of cell proliferation showed that the combined treatment of imatinib and Huaier extract exhibited a greater effect on cell inhibition. Using flow cytometry and Western blot, enhanced effects on the induction of cell apoptosis were observed. The combination of the two drugs also exhibited a significant effect in decreasing the protein and enzymatic activity levels of BCR-ABL. The molecular mechanisms may be involved in BCR-ABL related pathways, including the inactivation of p-AKT, p-STAT5, p-mTOR and p-Lyn. Consistent with the in vitro results, the combination of Huaier extract and imatinib inhibit the growth and infiltration of xenografted tumors. Taken together, our findings show that Huaier extract enhances the anticancer efficacy of imatinib in Ik6+ Ph+ ALL Further, it also provides a potential clinical application in the treatment of refractory Ph+ ALL.

Health-Related Quality of Life of Blinatumomab for Relapsed or Refractory B-Cell Precursor Acute Lymphoblastic Leukemia in a Randomized, Open-Label Phase 3 Study (TOWER): A Subgroup Analysis By Prior Allogeneic Hematopoietic Stem Cell Transplantation

Background In the phase 3 TOWER study, patients with relapsed or refractory (r/r) Ph– B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) who received blinatumomab had longer overall survival and improved health-related quality of life (HRQoL) versus patients who received standard of care chemotherapy (SOC). Because ALL patients who relapse from previous hematopoietic stem cell transplantation (HSCT) have poorer prognosis, we analyzed the HRQoL of TOWER patients with or without prior allogeneic HSCT (alloHSCT). Methods Adults (n=405) with r/r Ph– BCP ALL were randomized 2:1 to 2 cycles of induction blinatumomab (n=271) or SOC (n=134), followed by up to 3 consolidation cycles; 12 months of maintenance was allowed. AlloHSCT was allowed after cycle 1. HRQoL was assessed in cycle 1 (days 8, 15, and 29) using the EORTC QLQ-C30 Questionnaire. For global health status and functioning scales, a higher score indicates better HRQoL; for symptom scales/items, a lower score indicates better HRQoL. Time to deterioration analyses assessed the treatment effect based on a 10-point deterioration (clinically significant threshold) from baseline. Results Of 342 evaluable patients (blinatumomab, n=247; SOC, n=95); 114 (blinatumomab, n=85; SOC, n=29) and 228 (blinatumomab, n=162; SOC, n=66) did and did not, respectively, have prior alloHSCT. In those with no prior alloHSCT, changes in global health status were minimal for blinatumomab and SOC (Figure 1). In contrast, in patients with prior alloHSCT, global health status modestly improved in the blinatumomab group but worsened by ≥10 points at all time points for SOC, indicating clinically meaningful deterioration. Changes in functioning scales were minimal in the blinatumomab arm, both with or without prior alloHSCT, except for relatively improved emotional scores in patients with prior alloHSCT. In contrast, SOC patients had worsening in most functioning scales, regardless of prior alloHSCT status. This effect was most marked in those with prior alloHSCT, in whom physical, role, and social functioning deteriorated by ≥10 points. Similar patterns were observed for symptom scales/items: while blinatumomab was associated with improved or less worsening in symptom scores versus SOC, most symptom scores worsened in the SOC arm, especially in patients with prior alloHSCT, for whom all symptoms (except dyspnea) deteriorated by ≥10 points. Compared with SOC, blinatumomab was associated with a delayed time to clinically meaningful deterioration, particularly in patents with prior alloHSCT (Figure 2). Conclusions In patients with r/r Ph– BCP ALL, blinatumomab was associated with improved HRQoL compared with SOC, regardless of whether patients had received prior alloHSCT. Notably, however, the HRQoL benefit of blinatumomab was most pronounced among patients with prior alloHSCT.

Allogeneic CAR-T Cell Therapy for Treatment of Relapse after Allo-HSCT in Patients with Refractory CML Lymphoid Blast Crisis: Significance of HLA Matched Donor/Patient Pair in the Safety/Efficacy of CAR-T Cell Therapy

Introduction: CD19-specific CAR-T cells have shown promise in the treatment of relapsed or refractory Ph+ ALL. It remains to be established whether allogeneic CAR-T cells have clinical activity in patients with relapsed CML lymphoid blast crisis with a history of allo-HSCT. Here we report our experience in two cases of allogeneic CAR-T cell therapy for treatment of relapse after allo-HSCT in patients with refractory CML lymphoid blast crisis.Methods: For manufacture of allogeneic CAR-T cells, peripheral blood mononuclear cells were collected from the same stem cell donor. Lentiviral construction and generation of CAR-T cells, clinical protocol design, assessment and management of cytokine release syndrome (CRS), were performed as described in our previous report (Leukemia. 2017;31:2587-2593). Fludarabine and cyclophosphamide had been administered for lymphocyte depletion before allogeneic CAR-T cells infusion.Patients: Patient 1 was a 52-year-old woman with refractory CML lymphoid blast crisis, who had a relapse after undergoing allo-HSCT from her daughter (HLA-10/10). Her initial examinations of peripheral blood and bone marrow were consistent with the diagnosis of CML lymphoid blast crisis. Cytogenetics and molecular analysis confirmed the presence of t(9;22)(q34;q11) and BCR-ABL1 210 fusion protein. In February 2017, examination of bone marrow revealed a further increase of lymphoblasts to 83.2%. In addition, ABL1 kinase mutations (Y253H and E255K/V) were identified. The patient underwent HLA 10/10-matched allo-HSCT without acute GVHD. A remission with a negative test for BCR-ABL1 210 and 99.62% donor chimerism had been achieved, then she had a lymphoblastic relapse occurred 2 months after allo-HSCT. Consistently, BCR-ABL1 210 turned positive, and chimerism analysis showed 67.4% donor chimerism. 3 weeks after relapse, allogeneic CAR-T cells were infused at the dose of 5×106 /kg CD19-specific CAR-T cells.Patient 2 was a 39-year-old male patient with relapsed CML lymphoid blast crisis with a history of allo-HSCT. He had received a diagnosis of CML chronic phase 7 years earlier. Bone marrow revealed a karyotype of 46, XY, t(3;9;22)(q27;q34;q11) and BCR-ABL mRNA transcript. From April 2011 to September 2012, the patient was treated with nilotinib. In September 2012, bone marrow examination revealed 78% lymphoblasts, thus the diagnosis of CML lymphoid blast crisis was established. In December 2012, the patient underwent HLA 7/10-matched sibling allo-HSCT (from his brother) without evidence of GVHD and maintained CR for 2 years. In December 2014, the patient developed bone marrow relapse (lymphoblast 9.5%) and extramedullary leukemia (testicular involvement) harboring the BCR-ABL-T315I mutation. During 2014 to 2018, the patient received multiple courses of CIKs, HDMTX and DLI, but failed to achieve CR. In March 2018, the patient received healthy donor derived allogeneic CAR19 T cells (2×105/kg) therapy.Result: Before CAR-T cells infusion, both patients with refractory CML lymphoid blast crisis had a relapse after successful allo-HSCT. Approximately 1 month after CAR-T cells infusion, a persistent morphologic remission, a recovering BM, and complete absence of BCR-ABL mRNA transcripts confirmed morphologic and molecular remission in both patients. Consistent with this, flow cytometry could not detect blasts or CD19+ B lineage cells. Patient 1 did not experience toxicities and allogeneic CAR-T cell therapy was well tolerated. Patient 2 developed severe CRS (Gr 4) including high-grade fevers (>40°C), hypotension, hypoxia, mental status changes, and seizures. These episodes ran for approximately 1 week before they were halted by treatment with steroids plus tocilizumab, and plasma exchange. The toxicity of allogeneic CAR-T cells is correlated with high levels of IL-6, IFN-γ, TNF-a, and CRP.Conclusion: The clinical outcomes from these 2 patients demonstrate the in vivo efficacy of allogeneic CD19-targeted T cells to induce clinical, morphology and molecular remissions as well as B cell aplasia in adults with relapsed CML lymphoid blast crisis with a history of allo-HSCT. The efficacy of allogeneic CAR-T cell therapy may not always be related to the risk of severe CRS. The degree of HLA matching may have a major impact on the prevention of CRS after allogeneic CAR-T cell therapy. Fully HLA-matched-pair may increase the safety and efficacy of the allogeneic CAR-T cell therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5844: Targeting DNA repair in Philadelphia positive B-ALL

Abstract Introduction: The Philadelphia (Ph) chromosome was the first reported chromosomal rearrangement linked to a human malignancy. Ph-positive precursor B-cell acute lymphoblastic leukemic (Ph+Pre-B-ALL) expressing the BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis and high relapse rates in adults. BCR-ABL1 drives an aberrantly hyperactive DNA repair that confers survival advantages to leukemic cells and serves as its ‘Achilles' heel'. We used HDAC1,2 selective inhibitor in isolation or in combination with doxorubicin (a chemotherapy regimen component used for treating pre-B-ALL) to combat the aberrant DSB repair processes. Experimental design: We used a combination of molecular and cell biology, proteomics and functional genomics-based approaches and primary patient-derived xenograft mouse models to understand the genome stability mechanisms targeted by HDAC1,2 inhibition to overcome oncogenic signals promoted by the BCR-ABL1 fusion protein. Results: Mass-spectrometry analysis and DNA repair assays showed that HDAC1,2 inhibition and doxorubicin treatments impact common as well as distinct DNA repair and genome maintenance networks including decreased chromatin association of Mre11-Rad51-DNA ligase 1, the central hub of DNA repair. Translating this mechanistic knowledge, we then showed that the HDAC1,2 inhibitor either alone or when combined with a low dose doxorubicin caused cytotoxicity in primary patient Ph+ Pre-B-ALL cells, and decreased leukemia burden without adverse side-effects in vivo in refractory Ph+ Pre-B-ALL PDX models that we created. We recently found that HDAC1,2 targets H4K91ac mark in Ph+Pre-B-ALL cells. H4K91ac occurs within the body of nucleosome core and thus directly controls nucleosome or chromatin integrity. Increased H4K91ac following HDAC1,2 inhibition occurs around DSB sites genome-wide could cause a delay in the rate at which DSBs are repaired in Ph+Pre-B-ALL cells as a result of defective nucleosome structure around DSB sites. Conclusion: Our comprehensive study using a multipronged approach showed that HDAC1,2 inhibition as a monotherapy or in combination with a low-dose doxorubicin can override DSB repair ‘addiction' in Ph+ Pre-B-ALL. We are currently using the genome-wide ChIP-seq approach and an homing endonuclease DSB reporter system to examine how HDAC1,2 inhibition changes the chromatin landscape in Ph+Pre-B-ALL cells. Citation Format: Danielle Johnson, Shwetha Tharkar, Simon Jones, Matt Jarpe, Mahesh Chandrasekharan, Srividya Bhaskara. Targeting DNA repair in Philadelphia positive B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5844.

Phosphodiesterase-type 5 Inhibition for Refractory Pre-capillary PH in Advanced Heart Failure is Associated with Increased Early Mortality Post Heart Transplant

Phosphodiesterase-type 5 Inhibition for Refractory Pre-capillary PH in Advanced Heart Failure is Associated with Increased Early Mortality Post Heart Transplant

Abstract 2071: Sub-clonal heterogeneity and risk of treatment failure in Philadelphia-positive leukemias

Abstract Molecularly targeted therapies have been exceptionally successful in the treatment of some malignancies, most notably chronic myeloid leukemia (CML). However in other cancers, e.g. Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), drug resistance is a frequent occurrence. Resistance mutations may pre-exist as a consequence of intra-tumor heterogeneity; thus the extent of heterogeneity may determine the likelihood of treatment failure. Intra-tumor heterogeneity is challenging to quantitate, as heterogeneous mutations are likely to occur below the background error rate of conventional approaches for mutation detection. To overcome this limitation and explore the relationship between intra-tumor heterogeneity and drug resistance in Ph+ leukemias, we utilized single-molecule Duplex Sequencing (DS) of the ABL1 gene. DS eliminates sequencing errors by independently tagging the two strands of individual DNA molecules; true mutations are present at the same position in both strands, while PCR and sequencing errors are only present in one strand. DS has an error rate below 0.00001%, and thus enables quantitation of heterogeneous mutations with unprecedented resolution. We find that CP-CML, a disease that is often well controlled with targeted therapy, has an extremely low burden of intra-tumor heterogeneity at the time of diagnosis, with a sub-clonal mutation burden similar to that of normal individuals. Simulations which take into account the low cancer stem cell fraction of CP-CML and the sub-clonal mutation burden we measured reveal that only a minority of CP-CML patients will harbor pre-existing drug resistance, which may explain the unusual success of targeted therapy in this setting. Next, we studied patients with advanced Ph+ leukemias (refractory BP-CML and Ph+ ALL), which have a high rate of treatment failure and poor outcomes. We find that patients with BP-CML and Ph+ALL have a significant elevation in sub-clonal heterogeneity relative to those with CP-CML. We demonstrate that the higher extent of intra-tumor heterogeneity correlates with a significantly higher rate of drug resistance mutations and compound resistance (i.e. two concurrent resistance mutations) in patients. Unlike CP-CML, pre-existing mutations conferring resistance to targeted therapy are nearly certain in refractory BP-CML and Ph+ ALL as a consequence of the load of intra-tumor heterogeneity and high cancer stem cell fraction. We support this concept by three distinct approaches: (i) detection of pre-existing sub-clones that drive treatment failure; (ii) modeling the growth kinetics of resistant clones; and (iii) extrapolation of the mutation burden measured by DS. Our results suggest that intra-tumor heterogeneity influences the likelihood of resistance to molecularly targeted therapy. Quantitation of sub-clonal mutation load may be broadly applicable in other malignancies for predicting the likelihood of response to targeted therapy and monitoring disease progression. Citation Format: Michael W. Schmitt, Justin R. Pritchard, Lan Beppu, J. Graeme Hodgson, Victor M. Rivera, Lawrence A. Loeb, Jerald P. Radich. Sub-clonal heterogeneity and risk of treatment failure in Philadelphia-positive leukemias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2071. doi:10.1158/1538-7445.AM2017-2071

HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11–Rad51–DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair ‘addiction’ and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

Impact of early landmark responses with ponatinib on 4-yr outcomes in CP-CML patients (pts) in PACE, a pivotal phase II trial.

7050 Background: Ponatinib is approved for pts with refractory CML or Ph+ ALL for whom no other TKI therapy is indicated, or for patients with T315I. Previously (Mueller ASCO ‘16), we reported the positive association of early landmark responses with ponatinib on survival at 3 yrs in heavily pretreated pts with CP-CML in PACE (NCT01207440). Here, we provide an update with survival outcomes at 4 yrs. Methods: The association of molecular (assessed in a central lab) and cytogenetic responses (CyR) at 3, 6 and 12 mo with 4-yr post-landmark PFS and OS was evaluated in CP-CML pts (n = 267). P values: calculated using log-rank test. Data cutoff: 3 Oct ‘16. Results: At baseline, median time from diagnosis: 7 (range, 0.5–27) yrs; median age: 60 (18–94) yrs; median %Ph+: 100% (3–100), ≤10% Ph+: 19 pts (7%); 61% of pts had ≥3 prior TKIs. Among evaluable pts at 3, 6 and 12 mo, MCyR/CCyR was achieved in 48%/39%, 62%/52% and 71%/56% and MMR in 14%, 29% and 39% of pts, respectively. Greater reductions in BCR-ABL1 levels (Table) and CyR at most landmark time points were associated with improved 4-yr post-landmark PFS and OS. Deeper responses at all landmark time points were associated with achievement of MR4.5 over time. Conclusions: As with the 3-yr landmark analysis, CyR and deep reductions in BCR-ABL1 transcripts at early time points correlated with improved 4-yr post-landmark survival in this refractory population. These data continue to demonstrate the prognostic value of early cytogenetic and molecular responses with ponatinib in heavily pretreated pts with CP-CML. Clinical trial information: NCT01207440. [Table: see text]

Salvage therapy for children with relapsed or refractory Philadelphia chromosome-positive acute lymphoblastic leukemia.

In the tyrosine kinase inhibitor (TKI) era, outcomes after salvage therapy for relapsed or refractory Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) remain unclear. The clinical courses of 19 patients with relapse (n = 13) or induction failure (n = 6) in the Japanese Pediatric Leukemia/Lymphoma Study Group Ph+ ALL04 study were retrospectively reviewed. Fifteen male and four female patients had a median age of 8 (range 4-15) years at relapse or induction failure. Patients received imatinib in combination with hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-CVAD) and methotrexate and cytarabine (MTX/Ara-C) (n = 9), imatinib in combination with other chemotherapy (n = 5), chemotherapy without imatinib (n = 2), imatinib alone (n = 2), or no additional chemotherapy (n = 1). Two patients underwent hematopoietic stem cell transplantation (HSCT) without achieving complete remission (CR) and died of leukemia. The remaining 17 patients achieved CR with salvage therapies and underwent HSCT whilst in CR: 10 patients remain alive in CR, five died of transplantation-related complications, and two died of relapse. In six of seven patients with available data on minimal residual disease (MRD), imatinib in combination with the first course of hyper-CVAD was more effective in achieving a favorable MRD response compared with the Ph+ ALL04 induction regimen. This study suggested that cross-resistance to imatinib failed to develop after conventional chemotherapy. Imatinib in combination with chemotherapy including hyper-CVAD+MTX/Ara-C was effective and safe for relapsed or refractory Ph+ ALL patients who received frontline therapy without imatinib.

Analysis of the Sub-Clonal Origins of Compound Mutations in Patients with Refractory Ph+ Malignancies Treated with Ponatinib

Background: Ponatinib is a tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL1 and all single resistance mutants. In the Phase 2 PACE study (NCT01207440), ponatinib induced high rates of major hematological response (MaHR) and major cytogenetic response (MCyR) in BP-CML (31% and 23%) and Ph+ ALL (41% and 47%) patients (pts), even though 91% of pts had received at least 2 prior TKIs. However, the median progression free survival was only 4 months for BP-CML and 3 months for Ph+ ALL (Cortes JE, et al. N Engl J Med 2013). Previous studies have shown that certain compound mutations can cause resistance to ponatinib (Zabriskie et al. Cancer Cell 2014), but such mutations were found to be extremely rare in CP-CML pts from the PACE trial despite being heavily pre-treated (Deininger MW, et al. Blood 2016). Here we utilize a multi-level sequencing strategy that combines Sanger Sequencing (SS), Next Generation Sequencing (NGS), and single molecule Duplex Sequencing (DS), which is orders of magnitude more sensitive than NGS, to profile the mutational mechanisms that may account for the survival outcomes observed in BP-CML/Ph+ ALL pts treated with ponatinib in PACE.Methods: The BCR-ABL1 mutation status of the 267 CP-CML, 62 BP-CML, and 32 Ph+ ALL pts treated in the PACE trial was examined using SS and/or NGS as described previously (Cortes JE, et al. N Engl J Med 2013; Deininger MW, et al. Blood 2016). Mutations detected by SS at the end of treatment (EOT) but not at baseline (BL) were considered to have been gained during ponatinib treatment. DS was performed to determine whether mutations gained on treatment could be found in small sub-populations of cells at BL and to provide a direct measurement of the mutation burden in the ABL1 gene. Two distinct mathematical models were used to predict the size of pre-existing resistant sub-populations in BP-CML/Ph+ ALL pts based on the overall ABL1 mutation burden and the doubling time of clonal outgrowth.Results: The number of BCR-ABL1 mutations in BP-CML/Ph+ ALL patients at BL, as detected by SS, was not a significant predictor of intrinsic resistance to ponatinib in refractory patients with Odds Ratios (OR) of 1.0 (0.2-4.9 95% CI) for MaHR and 2.4 (0.5-12.5 95% CI) for MCyR. However, a larger proportion of BP-CML/Ph+ ALL pts, 30/61, acquired mutations in BCR-ABL1 at EOT vs. 19/130 CP-CML pts (p<0.005, OR 7.7 [1.5-53.3 95% CI]). Nearly all BP-CML/Ph+ ALL, but not CP-CML, pts who acquired mutations had BCR-ABL1 mutations at BL detected by SS: 27/30 vs. 10/19 CP-CML pts (p=8.5E-7, OR 5.6 [2.7-12.1 95% CI]). At least 19/27 BP-CML/Ph+ ALL pts had compound mutations at EOT, including 2 pts with 3 mutations in the same allele. Since the presence of pre-existing mutations from earlier TKI therapy is associated with further gain of mutations on ponatinib treatment, we used ultra-sensitive DS to determine if these resistant clones could be detected at BL. In 3/18 pts tested, the resistant clone was directly detected at BL in a small minority of cells. Mathematical modeling of leukemia growth rates in pts with mutation gains predicts that pre-treatment resistant population sizes range from ~1 cell to 2.7 x 108 cells in the peripheral blood, out of a total population of leukemic cells that ranged from ~1010 to 5 x 1011. Furthermore, DS and mathematical modeling allowed us to predict that many, if not all, documented resistance mutations were pre-existing in most refractory BP-CML/Ph+ ALL pts treated with ponatinib in the PACE trial.Conclusions: Clonal BL BCR-ABL1 mutation status determined by SS does not predict intrinsic resistance to ponatinib. However, in contrast to CP-CML, BP-CML/Ph+ ALL pts are more likely to relapse on ponatinib with acquired mutations in BCR-ABL1. Two distinct mathematical modeling strategies suggest that most, if not all, BCR-ABL1 resistance mutations in BP-CML/Ph+ ALL pts that emerge during ponatinib treatment were pre-existing. The majority of these pre-existing mutations were compound mutations whose evolutionary path was likely determined by prior TKI therapy. These findings emphasize the risk of the large population expansions that occur following prior TKI treatment failure and may explain the relatively high 2-yr event free survival rate of 81% observed in previously untreated Ph+ ALL pts treated with ponatinib (+hyper-CVAD) (Jabbour E, et al. Lancet Oncol 2015, Sasaki, et al. Cancer 2016).Study sponsor: ARIAD Pharmaceuticals, Inc. DisclosuresPritchard:ARIAD: Employment, Equity Ownership. Schmitt:TwinStrand: Equity Ownership. Hodgson:ARIAD: Employment, Equity Ownership. Lustgarten:ARIAD: Employment, Equity Ownership. Tang:ARIAD: Employment, Equity Ownership. Loeb:TwinStrand: Equity Ownership. Radich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; Pfizer: Consultancy; ARIAD: Consultancy; TwinStrand: Consultancy. Rivera:ARIAD: Employment, Equity Ownership.

Characteristics of ponatinib (PON) therapy for Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients (pts) in real-world (RW) clinical practice compared to the PACE trial.

e18514Background: The pivotal trial (PACE) studied the use of PON in adult pts with refractory Ph+ ALL and formed the basis of approval of PON. Prescribing data tracked by the specialty pharmacy ex...

Impact of landmark responses on 3-year (yr) outcomes with ponatinib in heavily pretreated CP-CML patients (pts).

7053Background: PON, a potent oral TKI, is active against native and mutant BCR-ABL1 and approved for pts with refractory CML or Ph+ALL, or with the T315I mutation. Pt responses at early landmark time points have been correlated with positive long-term (LT) outcomes in 1st and 2nd-lines. Here we evaluate the impact of early responses with PON on 3-yr outcomes in heavily pretreated pts (primarily ≥3rd line) with CP-CML in the ongoing Ph2 PACE trial. Methods: Molecular and cytogenetic responses at 3, 6 and 12 mo and their association with outcomes at 3 yrs were examined among CP-CML (n=267) pts in a posthoc analysis. P values: calculated using log-rank test. Data: as of 3 Aug 2015. Results: Median time from diagnosis was 7 yrs (range 0.5-27) and median age was 60 yrs (range 18-94); 61% had received ≥3 prior TKIs. At 3, 6, and 12 mo, 48%, 62% and 71% of pts, respectively, achieved MCyR, and 14%, 29% and 39%, respectively, achieved MMR, among pts evaluable at each landmark. Greater reductions in BCR-ABL1 tran...

Recommendations for utilization of the paracorporeal lung assist device in neonates and young children with pulmonary hypertension.

The management of decompensating critically ill children with severe PH is extremely challenging and requires a multidisciplinary approach. Unfortunately, even with optimal care, these children might continue to deteriorate and develop inadequate systemic perfusion and at times cardiac arrest secondary to a pulmonary hypertensive crisis. Tools to support these children are limited, and at times, the team should proceed with offering extracorporeal support, especially in newly diagnosed patients who have not benefitted from medical therapy prior to their acute deterioration, in patients with severe pulmonary venous disease and in patients with alveolar capillary dysplasia. Currently, the only approved mode for extracorporeal support in pediatric patients with PH eligible for lung transplantation is ECMO. To decrease the risks associated with ECMO, and offer potential for increased duration of support, extubation, and rehabilitation, we transitioned four small children with refractory PH from ECMO to a device comprising an oxygenator interposed between the PA and LA. This work describes in great detail our experience with this mode of support with emphasis on exclusion criteria, the implantation procedure, and the post-implantation management.