Interim Results of a Phase 1b/2 Study of Entospletinib (GS-9973) Monotherapy and in Combination with Chemotherapy in Patients with Acute Myeloid Leukemia

Abstract Background:Spleen tyrosine kinase (SYK) is a nonreceptor cytoplasmic tyrosine kinase primarily expressed in cells of hematopoietic lineage. Constitutive activation of SYK in acute myeloid leukemia (AML) has been reported and targeted inhibition of SYK induced differentiation in vitro and demonstrated anti-leukemia activity in AML mouse models. SYK has also been shown to directly phosphorylate the FLT3 receptor, modulating its activation and possibly promoting its role in leukemogenesis. Entospletinib is an orally bioavailable, selective inhibitor of SYK shown to be clinically active in B-cell malignancies. Here we evaluate the combination of entospletinib in patients with untreated AML using a 14-day window phase to assess single-agent activity, then adding standard intensive chemotherapy. Methods: In this phase 1b/2 study (NCT02343939), patients age 18 to 70 years with previously untreated AML, preserved organ function, and ECOG ≤ 2 were eligible to receive dose escalated entospletinib for 14 days as monotherapy (days -14 to 0) followed by combination with daunorubicin 60 mg/m2/d, cycle 1 day 1 to 3, and cytarabine 100 mg/m2/d, cycle 1 day 1 to 7. All patients received entospletinib monotherapy for up to 14 days prior to starting induction. Chemotherapy could be initiated after 5 days of monotherapy (and entospletinib continued for 4+ weeks) in patients with leukemia-related complications necessitating chemotherapy. Patients enrolled to dose level (DL) 0 and DL 1 received entospletinib 200 mg po BID and 400 mg po BID, respectively. Patients with residual disease two weeks after chemotherapy received a second induction cycle identical to the first. Entospletinib was continued without interruption until remission was assessed at count recovery. Results:Twelve patients enrolled with a median age of 54 (range, 18-69) years. Patients were in the following European LeukemiaNet genetic risk groups: favorable (n=1), intermediate I (n=3), intermediate II (n=2), and adverse (n=4), respectively. Three patients were not evaluable for dose limiting toxicity (DLT) assessment and were replaced (due to detection of CNS disease requiring non-study therapy (n=1), and withdrawal of consent unrelated to drug toxicity (n=2)). Single-agent entospletinib during the window period was well tolerated; toxicities after combination with intensive chemotherapy were common and typical. Among 3 patients treated at 200mg BID, no DLT was observed. Of 3 patients treated at 400mg BID, a patient with documented fungal pneumonia developed grade 3 pneumonitis that was possibly related to entospletinib. Although this did not meet DLT criteria, DL 1 was expanded with 3 additional patients, none of whom experienced DLT. Overall, the most common non hematologic adverse events (inclusive of intensive chemotherapy periods) were febrile neutropenia, nausea, and diarrhea. Based on this clinical experience and compiled pharmacokinetic data demonstrating lack of benefit to further dose escalation, 400 mg BID was selected as the recommended phase 2 dose. Responses were seen at both levels. Among the 3 patients treated at 200 mg BID, two required a second induction but all achieved a complete remission (CR) (3/3; 100%). Of the 6 patients treated at 400mg BID, none required a second induction and the CR rate was also 100%. Remarkably, an 18 year old male with 11q23-rearranged AML achieved morphologic and cytogenetic CR after only the 14 day entospletinib monotherapy window (prior to chemotherapy). Another patient with 11q23-rearranged AML had significant platelet response during the window period (this patient refused disease evaluation by marrow aspiration prior to chemotherapy). Conclusions: Entospletinib appears to have significant clinical activity in AML, and its combination at doses up to 400mg BID with intensive chemotherapy is well tolerated. An extended phase 2 program is now underway. Patients with 11q23-rearranged AML may be uniquely sensitive to SYK inhibition by entospletinib. Detailed molecular analysis of these patients is ongoing and will be presented. Disclosures Walker: Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding. Marcondes:Gilead Sciences: Employment, Equity Ownership. DiPaolo:Gilead Sciences: Employment, Equity Ownership. Abella-Dominicis:Gilead Sciences: Employment, Equity Ownership.

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EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114421 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1528-1528

Author(s):

Sebastian Stasik ◽

Jan Moritz Middeke ◽

Michael Kramer ◽

Christoph Rollig ◽

Alwin Krämer ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Mutational Status ◽

Equity Ownership ◽

Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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Sunitinib and Intensive Chemotherapy in Patients with Acute Myeloid Leukemia and Activating FLT3 Mutations: Results of the AMLSG 10-07 Study (ClinicalTrials.gov No. NCT00783653)

Blood ◽

10.1182/blood.v120.21.1483.1483 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1483-1483 ◽

Cited By ~ 1

Author(s):

Walter Fiedler ◽

Sabine Kayser ◽

Maxim Kebenko ◽

Jürgen Krauter ◽

Helmut R. Salih ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Induction Therapy ◽

Myeloid Leukemia ◽

Research Funding ◽

Single Agent ◽

Intensive Chemotherapy ◽

Consolidation Therapy ◽

Flt3 Mutations ◽

Level 1 ◽

Acute Myeloid

Abstract Abstract 1483 Background: Activating FLT3 mutations including internal tandem duplications (FLT3-ITD) and tyrosine-kinase domain mutation (FLT3-TKD) occur in approximately one third of patients with acute myeloid leukemia (AML) and are particularly associated with a poor outcome in case of FLT3-ITD. Sunitinib is a multitargeted FLT3 inhibitor approved for the treatment of advanced/metastatic renal cancer and metastatic/unresectable malignant GIST after failure of imatinib. Sunitinib has been evaluated in refractory AML as single agent treatment resulting in transient blast count reduction and in several cases of partial response in AML with activating FLT3 mutations. Aims: To evaluate the feasibility of a standard induction and consolidation therapy in combination with orally administered sunitinib in elderly AML patients with activating FLT3 mutations. Methods: Patients aged 60 years or higher with AML with activating FLT3 mutations (FLT3-ITD, FLT3-TKD) and fit enough for intensive chemotherapy were eligible. Induction therapy included cytarabine 100 mg/m2 per continuous infusion on days 1–7 and daunorubicin 60 mg/m2 i.v. on days 1–3 (DA). A second course was allowed in responding patients, who did not achieve a complete remission (CR). In patients achieving a CR after induction therapy three consolidation cycles were intended (cytarabine 1 g/m2 i.v. bid, on days 1,3,5). A 3+3 dose escalation/de-escalation scheme was used to define the dose and scheduling of sunitinib. The first cohort of three patients received oral sunitinib continuously starting from day 1 in a dose of 25 mg/day (level 1). Dose escalation to level 2 with sunitinib 37.5 mg/day continuously or dose de-escalation to level −1 with 25 mg day 1 to 7 had been defined in the protocol. After definition of the maximally tolerated dose (MTD) an extension of the cohort at that dose was intended. Results: A total of twenty-two patients were enrolled between January 2009 and March 2011. The median age was 70 years (range 60–78), 13 were female. The type of AML was de novo in 16 pts., s-AMLin one patient and t-AML in 4 pts. Fifteen patients had a FLT3-ITD (68%) and 7 a FLT3-TKD (32%) mutation. A NPM1 mutation was present in 11 patients (50%), 15 patients exhibited a normal karyotype, 3 an intermediate-2 risk karyotype according to ELN guidelines and 2 a complex karyotype and 2 had no evaluable metaphases. In the first cohort 5 patients were treated and two experienced dose-limiting toxicity (DLT), i) prolonged hematological recovery beyond day 35 in a patient achieving a CR and ii) a hand-foot-syndrome grade III. Four of the 5 patients achieved a CR. According to the protocol the following patients received treatment at dose level −1 with sunitinib 25mg days 1 to 7. In this cohort only one DLT occurred, again prolonged hematological recovery. Thus the MTD was defined at dose level −1. Response to induction therapy in all patients was CR in 13 pts. (59%), partial remission in 1 pt. (4.5%), refractory disease in 5 pts. (23%), death in 3 pts. (13.5%). CR rate in AML with FLT3-ITD was 53% (8/15) and 71% (5/7) in those with FLT3-TKD. All 13 patients achieving CR received repetitive cycles of high-dose cytarabine consolidation therapy and 7 proceeded to single agent sunitinib maintenance therapy (median 11 months, range 1–24 months). In these patients relapse occurred in 10, one patient died due to severe colitis during consolidation therapy and two patients are in sustained CR. Two patients not achieving a CR after induction therapy underwent allogeneic stem cell transplantation form matched unrelated donors. Twelve of the 22 patients died leading to a median survival of 18.8 months and a 2 year survival of 36% (95%-CI, 19–70%). Median relapse-free survival was 11 months. Conclusion: Combination of intensive induction and consolidation therapy with oral sunitinib in AML with activating FLT3 mutations is feasible with 25 mg sunitinib given during intensive therapy on days 1 to 7 and continuously during maintenance. Disclosures: Fiedler: Novartis: Consultancy, Research Funding; Pfizer Inc.: Consultancy, Research Funding.

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Chimeric Antigen Receptor-Modified T Cells for the Treatment of Acute Myeloid Leukemia Expressing CD33

Blood ◽

10.1182/blood.v128.22.4058.4058 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4058-4058 ◽

Cited By ~ 3

Author(s):

Degang Song ◽

Michael H. Swartz ◽

Steve G. Biesecker ◽

Fernando Borda ◽

Rutul R. Shah ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Chimeric Antigen Receptor ◽

Myeloid Leukemia ◽

Research Funding ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership ◽

Acute Myeloid

Abstract Relapsed acute myeloid leukemia (AML) is an aggressive disease with very poor outcomes. Redirection of T-cell specificity via chimeric antigen receptor (CAR) has shown promising anti-tumor activity in clinical trials, particularly for B cell linage malignancies. CD33 is a transmembrane protein expressed on normal and malignant myeloid-derived cells as well (as on subsets of activated T cells and NK cells). Since this protein is commonly expressed on AML cells, we sought to evaluate the efficacy of targeting AML with CD33-specific CAR-T cells. We generated a lentiviral construct to co-express CD33-specific CAR and a kill switch based on a tag derived from the epidermal growth factor receptor. The latter allows for the conditional elimination of CAR-T cells in vivo. Following transduction of primary T cells, we confirmed CAR and kill switch co-expression by flow cytometry and western blot analyses. Elimination of genetically modified T cells was demonstrated using the clinically-available antibody, cetuximab. CD33 CAR-T cells demonstrated specific cytotoxicity to CD33+ target cell lines. CD33 CAR-T cells were also activated to produce IFNg, TNF, and IL-2 cytokines in response to CD33+ target cells. Furthermore, adoptive transfer of CD33 CAR-T in immunocompromised (NSG) mice bearing established CD33+(CD19neg) AML (MOLM-13) tumor resulted in reduction of tumor burden and improvement of overall survival, compared to control mice receiving CD19 CAR-T cells or no immunotherapy (Figure). Sampling of blood demonstrated the persistence of the CD33 CAR-T cells with no detection of AML (MOLM-13) tumor cells. These pre-clinical data demonstrate the effectiveness of CD33 CAR-T cells in targeting CD33+ AML tumor cells and provide a rationale for future clinical evaluation in AML patients with unmet medical need. Disclosures Song: Intrexon Corporation: Employment, Equity Ownership. Swartz:Intrexon Corporation: Employment, Equity Ownership. Biesecker:Intrexon Corporation: Employment, Equity Ownership. Borda:Intrexon Corporation: Employment. Shah:Intrexon Corporation: Employment, Equity Ownership. Wierda:Genentech: Research Funding; Gilead: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding. Cooper:MD Anderson Cancer Center: Employment; Intrexon: Equity Ownership; Sangamo BioSciences: Patents & Royalties; Targazyme,Inc.,: Equity Ownership; City of Hope: Patents & Royalties; ZIOPHARM Oncology: Employment, Equity Ownership, Patents & Royalties; Miltenyi Biotec: Honoraria; Immatics: Equity Ownership. Chan:Intrexon Corporation: Employment, Equity Ownership.

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A Phase 2 Randomized Study of Low Dose Ara-C with or without Glasdegib (PF-04449913) in Untreated Patients with Acute Myeloid Leukemia or High-Risk Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v128.22.99.99 ◽

2016 ◽

Vol 128 (22) ◽

pp. 99-99 ◽

Cited By ~ 26

Author(s):

Jorge E. Cortes ◽

Florian H. Heidel ◽

Michael Heuser ◽

Walter Fiedler ◽

B. Douglas Smith ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

High Risk ◽

Myeloid Leukemia ◽

Research Funding ◽

Low Dose ◽

P Value ◽

Employment Equity ◽

Equity Ownership ◽

Acute Myeloid

Abstract Background: The Hedgehog signaling pathway (HhP) is aberrantly activated in leukemias and myelodysplastic syndrome (MDS), promoting cancer stem cell maintenance. HhP inhibition reduces leukemic stem cells. Glasdegib is a potent, selective, oral HhP inhibitor, with activity in pre-clinical and clinical studies. The addition of glasdegib to standard chemotherapy (CT) has an acceptable safety profile and appears to have clinical activity in MDS and acute myeloid leukemia (AML). Methods: In this study (NCT01546038), previously untreated AML or high-risk MDS patients (pts) ineligible for intensive CT were randomized 2:1 to receive low-dose cytarabine (LDAC) 20 mg subcutaneously twice a day x 10 days q28 days + oral glasdegib 100 mg daily or LDAC alone for as long as pts received clinical benefit. The primary endpoint was overall survival (OS). The final analysis was conducted after completion of recruitment (Oct 2015) and at least 92 OS events. Results: As of Apr 2016, 132 pts (116 AML, 16 MDS) were randomized to LDAC + glasdegib (n = 88) or LDAC alone (n = 44) (stratified as good/intermediate [int.] vs poor risk) (Table). Demographic and baseline characteristics were similar between arms in median age, baseline cytogenetic risk, and diagnosis. Eighty-four pts received LDAC + glasdegib and 41 pts LDAC alone (7 randomized/not treated pts were followed for survival). Median treatment duration was 83 days for LDAC + glasdegib and 47 days for LDAC alone; median follow up was 14.3 months and 12.4 months, respectively. In the glasdegib arm, 12 pts were continuing treatment and 25 were in follow up; in the LDAC arm, 1 pt was on treatment and 5 in follow up. Cytopenias and gastrointestinal toxicities were the adverse events (AEs) occurring more frequently in the LDAC + glasdegib arm. Hh-associated AEs in the glasdegib arm included dysgeusia (23.8%), muscle spasms (20.2%) and alopecia (10.7%). Serious AEs of febrile neutropenia were more frequent in the glasdegib arm, but sepsis rates were lower and pneumonia rates were similar. The most common cause of death was disease progression in both arms. Grade 2-4 QTcF prolongation was more frequent in the LDAC arm. Investigator-reported complete response (CR) rates were numerically higher for LDAC + glasdegib (n = 17, 15%) vs LDAC alone (n = 1, 2.3%), p-value 0.0142. Based on intent to treat analysis of 96 events, median OS (mOS) for LDAC + glasdegib was 8.3 (80% confidence interval [CI] 6.9, 9.9) vs 4.9 months (80% CI 3.5, 6.0) for LDAC alone (HR 0.511, 80% CI 0.386, 0.675; one-sided log rank p-value 0.0020 stratified by cytogenetic risk). For good/int. risk, mOS for LDAC + glasdegib was 12.2 vs 6.0 months for LDAC alone (HR 0.464, p-value 0.0035). For poor risk, mOS for LDAC + glasdegib was 4.4 vs 2.3 months (HR 0.575, p-value 0.0422). In AML pts, mOS for LDAC + glasdegib was 8.3 vs 4.3 months for LDAC alone (HR 0.462, p-value 0.0004). Conclusions: The addition of glasdegib to LDAC for AML and high-risk MDS pts improved OS compared with LDAC alone. The improvement was consistent among subgroups, particularly in good/int. risk pts. Treatment was associated with an acceptable safety profile. The addition of glasdegib to LDAC may be a treatment option for pts with AML or high-risk MDS. Disclosures Cortes: ARIAD: Consultancy, Research Funding; Bristol-Myers Squib: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. Heuser:Tetralogic: Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding; BerGenBio: Research Funding. Fiedler:Gilead: Other: Travel; Novartis: Consultancy; Ariad/Incyte: Consultancy; Teva: Other: Travel; Pfizer: Research Funding; Kolltan: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding; GSO: Other: Travel. Smith:Actinium Pharmaceuticals, Inc.: Research Funding. Robak:Pfizer: Research Funding. Montesinos Fernandez:Gamida Cell: Consultancy. Ma:Pfizer: Employment, Equity Ownership. Shaik:Pfizer: Employment, Equity Ownership. Zeremski:Pfizer: Employment, Equity Ownership. O'Connell:Pfizer: Employment, Equity Ownership. Chan:Pfizer: Employment, Equity Ownership.

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Concomitantly Targeting BCL-2 with Venetoclax (ABT-199/GDC-0199) and MAPK Signaling with Cobimetinib (GDC-0973) in Acute Myeloid Leukemia Models

Blood ◽

10.1182/blood.v126.23.2544.2544 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2544-2544 ◽

Cited By ~ 1

Author(s):

Lina Han ◽

Qi Zhang ◽

Ce Shi ◽

Joel Leverson ◽

Monique Dail ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Myeloid Leukemia ◽

Research Funding ◽

Genetic Alterations ◽

Leukemia Cells ◽

Intrinsic Resistance ◽

Employment Equity ◽

Equity Ownership ◽

Acute Myeloid

Abstract Pro-survival molecules including BCL-2 play critical roles in leukemia transformation and chemoresistance. ABT-199/GDC-0199 (venetoclax) is an orally available BH3-mimetic that binds with high affinity to BCL-2, but lacks affinity for BCL-XL and MCL-1. We have recently demonstrated anti-leukemia potency of venetoclax in acute myeloid leukemia (AML) models (Pan et al. Cancer Discovery 2014). However, venetoclax poorly inhibits MCL-1, causing resistance in leukemia cells that rely on MCL-1 for survival. The RAF/MEK/ERK (MAPK) cascade is a major effector pathway in AML that is activated by upstream mutant proteins such as FLT3, KIT and RAS. Additionally, the MAPK pathway regulates BCL-2 family proteins by stabilizing anti-apoptotic MCL-1 and inactivating pro-apoptotic BIM. In this study, we evaluated the anti-tumor effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib).. We initially examined activity of these agents in a panel of myeloid leukemia cell lines with diverse genetic alterations (Fig. 1A). The IC50 values of cobimetinib ranged from < 0.01 µM to > 1 µM after 72 hours of drug treatment but did not correlate with the basal level of p-ERK1/2. In 7 out of 11 cell lines, combination of the agents elicited synergistic growth inhibition. Notably synergism of venetoclax with cobimetinib was observed in venetoclax-resistant cell lines (MOLM14, OCI-AML3, NB4 and THP1). Ongoing analysis of pharmacodynamic markers include transcriptome assessment by RNA sequencing, functional proteomics by reverse phase protein array (RPPA) and quantification of BCL-2:BIM and MCL-1:BIM complexes using the electrochemiluminescent ELISA assay (Meso Scale Discovery, MSD-ELISA). The preliminary MSD data revealed that BCL-2:BIM complex was disrupted in most cell lines and accumulated following cobimetinib treatment, which may be due to the disruption of MCL-1:BIM complex by inhibition of MEK (Fig. 1B). In a long-term culture of primary AML blasts in serum-free stem cell growth medium supplemented with cytokines and StemRegenin 1 (SR1) to main the immature state of leukemia cells, the combination of venetoclax and cobimetinib induced distinct apoptotic cell death, with AML #1 sensitive to venetoclax but resistant to cobimetinib. Alternatively, AML #2 and #3 samples were resistant to venetoclax but sensitive to cobimetinib and the combination of both drugs (Fig. 1C). We next investigated signaling patterns and BCL-2 family protein expression in AML stem/progenitor cells using a 34-antibody panel and time-of-flight mass cytometry (CyTOF). In AML#1, BCL-2 was expressed in leukemia blasts, with enrichment in a progenitor AML population phenotypically defined as CD45dim CD34+ CD38+ CD123+ CD33+ (Fig. 1D). The high expression level of BCL-2 and low expression of MCL-1 and BCL-XL may account for sensitivity to venetoclax in AML#1. Both basal and G-CSF- or SCF-stimulated p-ERK was efficiently down-regulated by cobimetinib; however, G-CSF-evoked p-STAT3/5 and SCF-induced p-AKT were only slightly reduced (Fig. 1E). Notably we observed increased phosphorylation of STAT5 pathway upon treatment with cobimetinib, suggesting that active MAPK signals inhibit phosphorylation of the JAK-STAT pathway, as previously reported (Krasilnikov et al. Oncogene, 2003 and Lee at al. Cancer Cell, 2014). To test the efficacy of both compounds in vivo, we injected NSG mice with genetically engineered OCI-AML3/Luc/GFP cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the cobimetinib single agent and venetoclax plus cobimetinib co-treated mice (Fig. 1F). The efficacy study is ongoing and median survival for cobimetinib and venetoclax co-treated mice has yet to be determined (Fig. 1G). In summary, our data demonstrates that combinatorial blockade of MAPK and BCL-2 pathways is synergistic in the majority of AML cell lines tested and can overcome intrinsic resistance to venetoclax. Ongoing studies will evaluate efficacy of this combination therapy in primary human AML xenografts and elucidate mechanisms of synergy. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Dail:Genentech: Employment, Equity Ownership. Phillips:AbbVie: Employment, Other: Shareholder, Patents & Royalties. Chen:Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Jabbour:Pfizer: Consultancy, Research Funding. Sampath:Genentech: Employment, Equity Ownership. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

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Combining p38MAPK Inhibitors with a Second Targeted Agent Enhances Blockade of Inflammatory Signaling-Mediated Survival in Acute Myeloid Leukemia Cells

Blood ◽

10.1182/blood-2018-99-117537 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2726-2726

Author(s):

Stephen E Kurtz ◽

Christopher A. Eide ◽

Narain P. Dubey ◽

Andy Kaempf ◽

Shannon K. McWeeney ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Single Agent ◽

Combination Drug ◽

Advisory Committees ◽

Ic50 Values ◽

Equity Ownership ◽

Acute Myeloid

Abstract Background : Various pro-inflammatory and stress-related stimuli activate p38 mitogen-activated protein kinase (p38MAPK), triggering cascades of cell proliferation, differentiation, and apoptosis signaling. We have previously found that inflammatory cytokines promote growth and survival in primary cells from acute myeloid leukemia (AML) patients. The downstream mediator of inflammatory pathways is p38MAPK, and blocking this regulator with kinase inhibitors abrogates inflammation signaling in AML cells. Methods : We used a functional ex vivo screening assay to identify small-molecule targeted inhibitors and inhibitor combinations demonstrating selective efficacy across broad categories of leukemia. Primary mononuclear cells isolated from leukemia patients (n=408) were plated in the presence of the p38MAPK inhibitor doramapimod or combinations of doramapimod with a second targeted agent that inhibits or effects a non-overlapping biological pathway. A 7-point concentration series was evaluated for both single agent inhibitors and combinations. Leukemia specimens from 408 unique patients were classified into 5 diagnosis subgroups including acute myeloid leukemia (AML; n=206), acute lymphoblastic leukemia (ALL; n=42), chronic lymphocytic leukemia (CLL; n=115), chronic myeloid leukemia (CML; n=16) and myeloproliferative neoplasms/myelodysplastic syndrome (MPN or MDS/MPN; n=29). IC50 and AUC values were derived from probit-based regression for each response curve. Mutational status for FLT3-ITD and NPM1 were compiled from clinical labs or by capillary electrophoresis using a QiaXcel instrument. Disease status was obtained from clinical chart review. For a subset of AML samples, RNAseq analysis was obtained in parallel to drug sensitivity testing. Single and combination drug treatment IC50 and AUC values were compared within groups by Friedman test, across groups by Kruskal-Wallis test, and with continuous variables by Spearman rank correlation. Results : Among diagnostic subgroups tested, AML specimens showed the lowest median IC50 for doramapimod (1.71 uM), with 75 of 206 samples tested (36%) exhibiting IC50 values < 0.5 uM. In contrast, CLL specimens were significantly less sensitive (median IC50: 4.73 uM), with 27 of 115 samples tested (23%) showing IC50< 0.5 uM. For ALL, CML, and MDS/MPN subgroups, median IC50 values were 10, 3.19, and 6.78 uM, respectively; this translated to 17% of ALL, 19% of CML, and 38% of MDS/MPN samples with IC50< 0.5 uM. Doramapimod was also tested in combination with inhibitors of histone deacetylase (panobinostat), cyclin-dependent kinases 4/6 (CDK4/6; palbociclib), bromodomain and extra-terminal (BET) domain (JQ1), and BCL2 (venetoclax). Doramapimod combinations with panobinostat or JQ1 did not show enhanced efficacy compared to either single agent. However, combining doramapimod with palbociclib or venetoclax resulted in significantly enhanced efficacy compared to each single agent (median IC50: 0.014 and 0.075 uM, respectively; p<0.0001). Similar results were obtained using AUC as a drug effect measure. Of the 206 AML samples tested, 75% were sensitive (IC50 values < 0.5 uM) to both of these combinations, whereas 8% and 14% were sensitive to doramapimod in combination with only palbociclib or venetoclax, respectively. Sensitivity to combinations of doramapimod and palbociclib or venetoclax was not significantly associated with either age or gender. For the doramapimod + palbociclib combination, drug sensitivity (AUC) was correlated with gene expression for p38MAPKδ(Spearman r: -0.25). For doramapimod + venetoclax, sensitivity was correlated with expression of BCL2, MCL1, p38MAPKγ (Spearman r: -0.53, 0.23, and -0.33, respectively). Further analysis to align drug sensitivities with additional clinical and genetic features and inflammatory gene expression for AML patient samples is in progress. Conclusions : AML patient specimens demonstrate ex vivo sensitivity to inhibition of p38MAPK, and this efficacy is enhanced when combined with inhibitors of either CDK4/6 or BCL2, suggesting dual inhibition of these pathways may extend clinical utility among patients with genetically heterogeneous leukemia. Disclosures Druker: Celgene: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Millipore: Patents & Royalties; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Fred Hutchinson Cancer Research Center: Research Funding; Monojul: Consultancy; ARIAD: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy. Tyner:Genentech: Research Funding; Aptose: Research Funding; Janssen: Research Funding; Array: Research Funding; Incyte: Research Funding; Takeda: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees.

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Integrated Transcriptomic and Genomic Sequencing Identifies Prognostic Constellations of Driver Mutations in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Blood ◽

10.1182/blood-2019-132746 ◽

2019 ◽

Vol 134 (Supplement_2) ◽

pp. LBA-4-LBA-4 ◽

Cited By ~ 1

Author(s):

Ilaria Iacobucci ◽

Manja Meggendorfer ◽

Niroshan Nadarajah ◽

Stanley Pounds ◽

Lei Shi ◽

...

Keyword(s):

Gene Expression ◽

Acute Myeloid Leukemia ◽

Transcriptome Sequencing ◽

Myeloid Leukemia ◽

Research Funding ◽

Genomic Sequencing ◽

Expression Data ◽

Employment Equity ◽

Equity Ownership ◽

Acute Myeloid

CG Mullighan and T Haferlach: are co-senior authors Introduction: Recent genomic sequencing studies have advanced our understanding of the pathogenesis of myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), and improved classification of specific subgroups. Unfortunately, these studies have mostly analyzed specific subtypes and/or used targeted DNA-sequencing, thus limiting discovery of novel mutational patterns and gene expression clusters. Here, we performed an integrated genome-wide mutational/transcriptomic analysis of a large cohort of adult AML and MDS samples to accurately define subtypes of diagnostic, prognostic and therapeutic relevance. Methods: We performed unbiased whole genome (WGS) and transcriptome sequencing (RNA-seq) of 1,304 adult individuals (598 AML and 706 MDS; Fig. 1A), incorporating analysis of somatic and presumed germline sequence mutations, chimeric fusions and structural complex variations. Transcriptomic gene expression data were processed by a rigorous bootstrap procedure to define gene expression subgroups in an unsupervised manner. Associations between genetic variants, gene expression groups and outcome were examined. Results: Genomic/transcriptome sequencing confirmed diagnosis according to WHO 2016 of AML with recurrent genetic abnormalities in 10.9% of cases. These cases had a distinct gene expression profile (Fig. 1A), good prognosis (Fig. 1B) and a combination of mutations in the following genes: KIT, ZBTB7A, ASXL2, RAD21, CSF3R and DNM2 in RUNX1-RUNXT1 leukemia; FLT3, DDX54, WT1 and CALR in PML-RARA promyelocytic leukemia; KIT and BCORL1 in CBFB-rearranged leukemia. In addition, 9% of cases showed rearrangements of KMT2A, with known (e.g. MLLT3) and non-canonical partners (e.g. ACACA, and NCBP1) and poor outcome. Although common targets of mutations have been previously described for myeloid malignancies, the heterogeneity and complexity of mutational patterns, their expression signature and outcome here described are novel. Gene expression analysis identified groups of AML and/or MDS lacking recurrent cytogenetic abnormalities (87%). The spectrum of the most frequently mutated genes (>10 cases) and associated gene expression subtypes is summarized in Figure 1A. TET2 (more frequent in MDS than AML, p=0.0011) and DNMT3A (more frequent in AML than MDS, p<0.0001) were the most frequently mutated genes. Interestingly, mutations in these genes promoting clonal hematopoiesis were significantly enriched in the subgroup with NPM1 mutations. Overall, NPM1 mutations occurred in 27.4% of AML and 1% of MDS and were characterized by four expression signatures with different combination of cooperating mutations in cohesin and signaling genes and outcome (Fig. 1C, gene expression, GE, groups 2, 3, 7 and 8). Co-occurring NPM1 and FLT3 mutations conferred poorer outcome compared to only NPM1, in contrast co-occurring mutations with cohesin genes had better outcome (Fig. 1D). Additional mutations that significantly co-occurred with NPM1 were in PTPN11, IDH1/2, RAD21 and SMC1A. Three gene expression clusters accounted for additional 9% of cases with mutual exclusive mutations in RUNX1,TP53 and CEBPA and co-occurring with a combination of mutations in DNA methylation, splicing and signaling genes (Fig. 1E, GE groups 4, 5 and 6). Interestingly, RUNX1 mutations were significantly associated with SRSF2 mutations but not with SF3B1, showed high expression of MN1 and poor outcome (Fig. 1F). In contrast to the distinct, mutation-associated patterns of gene expression in AML samples, the gene expression profile of MDS was less variable despite diversity in patterns of mutation. MDS was enriched in mutations of SF3B1 (27.2%), mutually exclusive with SFRS2 (14.4%) and U2AF1 (5.5%); TP53 (13.7%) and RUNX1 (10.5%) and a combination of mutations in epigenetic regulators with outcome dependent on mutational pattern (Fig. 1A, G-H). Moreover, structural variations and/or missense mutations of MECOM accounted for 2% of cases. Conclusions: the integration of mutational and expression data from a large cohort of adult pan myeloid leukemia cases enabled the definition of subtypes and constellations of mutations and have prognostic significance that transcends prior gene panel-based classification schema. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Clinical Characteristics and Outcome in IDH1/2 Mutant AML Patients - Analysis of 3898 Newly Diagnosed Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-118128 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1461-1461 ◽

Cited By ~ 2

Author(s):

Jan Moritz Middeke ◽

Christoph Rollig ◽

Michael Kramer ◽

Alwin Kramer ◽

Tilman Bochtler ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Normal Karyotype ◽

Newly Diagnosed ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Acute Myeloid

Abstract Purpose Mutations of the isocitrate dehydrogenase-1 (IDH1) and IDH2 genes are one of the most frequent alterations in acute myeloid leukemia (AML) and can be found in ~20% of patients at diagnosis. Several IDH inhibitors are currently in late stage clinical development with Enasidenib, an IDH2 inhibitor, being recently approved by the FDA. Previous analyses have reported differential impact on response to chemotherapy and outcome, depending on the IDH-mutation type, co-occurring mutations and cytogenetic abnormalities, as well as the variant allele frequency (VAF) of IDH mutations. In order to better understand its prognostic role, we analyzed newly diagnosed AML patients enrolled in prospective trials of the Study Alliance Leukemia (SAL) to investigate the impact of IDH1/2 mutations on outcome. Patients and Methods All AML patients consecutively enrolled into intensive AML treatment protocols of the SAL or into the SAL registry were included in this analysis. Next-generation sequencing (NGS) on an Illumina MiSeq-system was performed to detect IDH1/2 mutations using pre-treatment samples. Overall survival (OS) and response to therapy were analyzed for all patients with intensive treatment and according to the mutational status. Results Overall, samples of 3898 patients were analyzed. The median follow-up was 91 months (95% CI 87.2 - 93.9). Patients' characteristics are shown in Tbl.1. Three-hundred twenty-nine patients (8.4%) had IDH1 mutations and 423 (11%) had IDH2 mutations; both mutations were found in 12 pts, so the overall mutation rate in IDH1 and 2 was 19% (740/3898 patients). Of the IDH1 variants, the most common ones were the R132C found in 143 patients (43%) and R132H in 137 patients (42%). For IDH2, 324 patients had the R140Q (77%) and 80 patients the R172K (19%) variant. According to the two main variants of the more common IDH2 mutations, as reported before, the IDH2 R172K was mutually exclusive with NPM1 and/or FLT3-ITD mutations. Overall, there was a trend for increased OS for patients with IDH2 R172K (26 vs. 15 months) as compared to those with R140Q. Considering only patients with a normal karyotype and no NPM1/FLT3-ITD mutation, these patients (n=27) had a highly significant better OS than patients with IDH2 R140Q (46.3 vs. 13.1 months, p=.012), supporting the findings published by Papaemmanuil et al. (NEJM 2016). In IDH1-mutated patients, we observed statistically significant differences in baseline characteristics between the two most common mutation types, IDH1 R132C and R132H. Patients carrying the R132C mutation were older (62 vs. 55 years, p=.001), had lower WBC (3.6 vs. 21 Gpt/L, p≤.001) and were less likely to have a normal karyotype (43% vs. 66%, p=.002), NPM1 (23% vs. 66%, p=<.001), and FLT3-ITD mutations (8% vs. 27%, p<.001) than those with the R132H variant. In univariate testing, the CR rate was also statistically significant lower in patients with IDH1 R132C (53% vs. 72%, p≤.001), with a median OS of 12.9 months compared to 17.4 months for patients with R132H variant (p=.08). In multivariate analysis including age, WBC, NPM1 and FLT3 status, and ELN risk, the CR rate was significantly lower in patients with the IDH1 R132C variant (p=.038). The median IDH VAF was 38% (range, 0.1 - 58) with no difference according to the different types of mutation. Patients with a VAF > 30% had a significantly higher BM blast count (73% vs 40% for VAF≤5%) and WBC (21.2 Gpt/L vs. 3.7 Gpt/L) at baseline, but there was no clear impact on CR rate or OS found in multivariate analysis. Conclusion In this large cohort of AML patients with IDH1/2 mutations, we found significant and so far not reported differences for one of the two most prominent mutations types of IDH1. The R132C variant was associated with increased age, lower WBC, and lower NPM1 and/or FLT3 co-mutation rate. Further, these patients had lower CR rates and a trend for shorter OS. For IDH2 we were able to reproduce findings on co-mutations and showed a favorable outcome for intensively treated patients with a normal karyotype and no NPM1/FLT3-ITD mutation and the IDH2 R172K variant, providing additional evidence for classification as a separate AML entity. Disclosures Middeke: Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Kramer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Daiichi Sankyo: Consultancy. Scholl:Alexion: Other: Travel support; Abbivie: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; MDS: Other: Travel support; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Incyte: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Brümmendorf:Takeda: Consultancy; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Novartis: Consultancy, Research Funding. Burchert:Novartis: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Takeda: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Johnson & Johnson: Research Funding; Roche: Research Funding; Eisai: Research Funding; Affimed: Research Funding; Novartis: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Schetelig:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Janssen: Consultancy, Honoraria; Roche: Honoraria; Sanofi: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Stoelzel:Neovii: Speakers Bureau.

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Clonal Heterogeneity in Differentiation Response and Resistance to the IDH2 Inhibitor Enasidenib in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.724.724 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 724-724

Author(s):

Lynn Quek ◽

Muriel David ◽

Alison Kennedy ◽

Marlen Metzner ◽

Michael Amatangelo ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Single Cell ◽

Myeloid Leukemia ◽

Research Funding ◽

Functional Heterogeneity ◽

Employment Equity ◽

Sequential Samples ◽

Equity Ownership ◽

Acute Myeloid ◽

Refractory Aml

Abstract Background Mutations in Isocitrate Dehydrogenase 2 (IDH2) occur in many cancers including Acute Myeloid Leukemia (AML). In preclinical models mutant IDH2 (mIDH2) causes partial hemopoietic differentiation block1. Recently, we showed that single agent enasidenib, a first-in-class, selective mIDH2 inhibitor, produces a 40% response in relapsed/refractory AML patients by promoting differentiation2. Here, we studied response and acquired resistance to enasidenib, in sequential samples treated in the Phase 1 study of Enasidenib in relapsed/ refractory AML patients. Results We studied a cytogenetically and genetically representative subset of 25 patients enriched for enasidenib responders, genotyped by whole exome sequencing (WES) or cancer gene panel targeted re-sequencing. Pre-enasidenib, differentiation arrest in these AML patients resulted in abnormally expanded leukaemic progenitors or precursors and diminished mature haematopoietic populations. Complete remission (CR) post-enasidenib was associated with in increased mature populations, near-normalisation of haematopoietic progenitor profiles, and restoration of in vitro progenitor function. In most patients, mature blood cells (of erythroid and granulocyte-monocyte lineages) post-enasidenib are IDH2 mutant, consistent with enasidenib inducing differentiation of IDH2 mutant leukaemic progenitors/ precursors. Each mIDH2 patient studied had on average 13 somatic, non-synonymous exonic or splice site mutations in addition to IDH2 . We used single cell genotyping (SCG) to reveal linear or branching clonal structures in mIDH2 AML. We combined clonal structure data and immunophenotyping of haematopoietic progenitor, precursor and mature populations to track functional behaviour of mIDH2 clones before, and during enasidenib treatment. We demonstrate, for the first time, that mIDH2 subclones within the same patient are functionally heterogeneous: both in their ability to differentiate pre-enasidenib, and in their sensitivity to Enasidenib-induced differentiation. This suggests that different combinations of co-operating mutations result in functional heterogeneity of mIDH2 clones. When we studied the contribution of mIDH2 clones to functional haematopoiesis at CR, we found that this was supported by either ancestral or leukaemic terminal mIDH2 clones. Despite a median survival of 18-21 months in patients who respond to enasidenib, most patients eventually relapse3. In contrast to targeted therapies such as tyrosine kinase inhibitors, in all 12 relapse samples studied, none had second site mutations in IDH2 . Furthermore, 2-hydroxyglurate (2HG) levels remain suppressed in most patients suggesting enasidenib remains effective in inhibiting mIDH2 enzyme. Instead, mIDH2 clones, which had persisted at CR or partial remission (PR) acquired additional mutations or aneuploidy, highlighting bypass pathways which re-impose differentiation arrest. We found 4 patterns: i) acquisition of IDH1 codon R132 mutations which resulted in a rise in 2HG (n=2), ii) deletion of chromosome 7q (n=4), iii) gain of function mutations in genes implicated in cell proliferation (FLT3, CSF3R) (n=3) and iv) mutation in hematopoietic transcription factors (GATA2, RUNX1) (n=2). We also found mutations in 4 genes (DHX15 and DEAF1 (n=1) ; NFKB1 (n=1) and MTUS1 (n=1)) not previously implicated in haematopoietic differentiation arrest which were selected for, or evolved in mIDH2 subclones at relapse. Conclusion This study provides a paradigm of how deep clonal single cell analysis in purified hemopoietic compartments in sequential samples through therapy reveals clonal complexity and the impact of the selective pressure of therapy on clonal architecture. Furthermore, we gain insights into the functional heterogeneity of mIDH2 subclones in their ability to differentiate pre-and post-Enasidenib. Further analysis of this kind in a larger cohort of IDH2 -inhibitor-treated patients would also provide insight to improve efficacy of this novel class of therapeutics, and design of combination therapies in AML and other cancers. Finally, this provides a platform for further study of the pathways mediating enasidenib resistance. References 1. Kats, L.M. , et al. Cell Stem Cell14, 329-341 (2014). 2. Amatangelo, M.D. , et al. Blood (2017). 3. Stein, E.M. , et al. Blood (2017). Disclosures Quek: Celgene Corporation: Research Funding. Amatangelo: Celgene Corporation: Employment. Agresta: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yen: Agios: Employment, Equity Ownership. Stein: Pfizer: Consultancy, Other: Travel expenses; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Constellation Pharma: Research Funding; Novartis: Consultancy, Research Funding; GSK: Other: Advisory Board, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding. De Botton: Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria. Thakurta: Celgene Corporation: Employment, Equity Ownership. Levine: Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding. Vyas: Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

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Preliminary Results of a Phase 1 Study of Flotetuzumab, a CD123 x CD3 Bispecific Dart® Protein, in Patients with Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v130.suppl_1.637.637 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 637-637

Author(s):

Geoffrey L. Uy ◽

John Godwin ◽

Michael P Rettig ◽

Norbert Vey ◽

Matthew Foster ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Research Funding ◽

Phase 1 ◽

Target Dose ◽

Employment Equity ◽

Cytokine Levels ◽

One Step ◽

Equity Ownership ◽

Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) blast and leukemic stem cells highly express CD123, which is associated with high-risk disease and disease progression. CD123 expression on normal hematopoietic stem cells is minimal, enabling a strategy of preferential ablation of AML with a CD123-targeted approach. Flotetuzumab (MGD006/S80880) is a novel T-cell redirecting (CD123 x CD3) bispecific DART® protein being tested in a phase 1 study in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: This phase 1 dose-escalation study is designed to define the safety profile, maximum tolerated dose and schedule (MTDS), and preliminary anti-leukemic activity of flotetuzumab. Relapsed/refractory (R/R) AML or intermediate-2/high-risk MDS patients (pts) are treated on 28-day cycles at doses from 3-1000ng/kg/day on one of 2 dosing schedules (4-day on/3-day off or a continuous 7-day on schedule). To mitigate cytokine-release syndrome (CRS), a one-step lead-in dose (LID) (100ng/kg/day for 4 days) or two-step LID (30 ng/kg/day for 3 days followed by 100ng/kg/day for 4 days) was instituted during Cycle 1/Week 1 (C1W1), followed by the cohort target dose (300-1000ng/kg/day) on either of the dosing schedules on W2-4. Cycle 2 and beyond, all pts were treated on a 4-day on/3-day off schedule at the cohort target dose for a maximum of 12 cycles, with 2 cycles after a complete response or complete remission with incomplete blood count recovery. Steroid-sparing, anti-cytokine therapy was used, if clinically indicated, to manage CRS symptoms. Disease status was assessed by International Working Group (IWG) criteria. Samples were collected for pharmacokinetics, anti-drug antibodies (ADA) and cytokine analysis, including IL-2, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma and GM-CSF. Results: 45 pts with R/R AML/MDS (89% AML and 11% MDS) have been treated with flotetuzumab, median age of 64 (29-84), and 44% female. The MTDS has been reached at 500ng/kg/day. Overall flotetuzumab has demonstrated manageable toxicity; drug-related adverse events ≥G3 were observed in 20/45 (44%) pts; infusion-related reaction/CRS (IRR/CRS), the most common toxicity, was observed in 34/45 (76%) pts (G3 in 6/45, 13%). The most frequent CRS symptoms were pyrexia (15), chills (10), tachycardia (10), and hypotension (4). Cytokine levels were higher in pts with CRS than in pts without (median IL-6, 116.2 vs. 67.9 pg/mL; IL-8, 191.1 vs. 144.6 pg/mL; IL-10, 867.6 vs. 348.7 pg/mL) and were generally higher with increasing CRS grade. A two-step LID during week 1 appeared to decrease the severity of CRS grade (mean grade reduction 0.54) compared to pts that received a one-step LID during cycle 1. In addition, lower median peak cytokine levels are observed with two-step LID during W1 and after achieving W2 target dose. Fourteen pts treated at the threshold 500 ng/kg/day dose cohort and beyond (700ng/kg/day dose cohort) have completed at least one cycle of treatment and had a post-treatment bone marrow (BM) biopsy. Anti-leukemic activity was documented in 57% (8/14) pts, 6/14 reached IWG criteria (3 CR, 1 CRi, 1 MLF, 1 PR) for an overall response rate (ORR) of 43%, and 2 pts had stable disease and bone marrow (BM) blast reduction of 20 and 25% from baseline, respectively. Blast reduction occurred rapidly, often within one cycle of therapy, and extended beyond flotetuzumab discontinuation. Multispectral immunohistochemistry analysis of BM samples showed flotetuzumab in situ with a significant increase (in CD-8 T cells (1.58-fold increase, p=0.0013). Consistent with T-cell activation, CD-25, CD-69 and PD-1 upregulation on both CD-4 and CD-8 T-cells was also observed in peripheral blood samples. Conclusions: Flotetuzumab in R/R AML and MDS demonstrated evidence of anti-leukemic activity (ORR 43%) with a manageable safety profile. This program advances an immune-activating agent in treating AML and continues to enroll patients in cohort expansion (24 AML and 24 MDS patients at the MTDS) in the US and Europe. clinicaltrials.gov NCT02152956. Disclosures Uy: Boehringer Ingelheim: Consultancy; GlycoMimetics: Consultancy; Novartis: Consultancy, Other: Travel Suppport. Foster: Macrogenics: Research Funding; Shire: Honoraria; Pfizer: Research Funding; Amgen: Honoraria; Incyte: Honoraria; Celgene: Research Funding; Celator: Research Funding. Arellano: Cephalon Oncology: Research Funding. Rizzieri: Shire: Research Funding; Erytech: Research Funding. Topp: Roche: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Research Funding; Macrogenics: Consultancy, Research Funding; Celgene: Other: Travel; Regeneron: Consultancy, Honoraria, Research Funding. Martinelli: Incyte, Pfizer, MSD, Abbvie, J&J, Seattle Genetics, Jazz Pharmaceuticals, Astellas: Consultancy, Other: Advisory Board, Speakers Bureau. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Lelièvre: Institut de recherches international Servier: Employment. La Motte-Mohs: Sunnybrook Health Sciences Centre: Patents & Royalties; MacroGenics: Employment, Equity Ownership, Patents & Royalties. Sun: Macrogenics Inc: Employment, Equity Ownership. Baughman: MacroGenics, Inc.: Employment. Shannon: MacroGenics, Inc.: Employment. Fox: Bristol Myers-Squibb: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy, Research Funding; PerkinElmer: Consultancy, Research Funding; Janssen/Johnson and Johnson: Consultancy, Research Funding; Argos: Consultancy; Bayer: Consultancy; Definiens: Consultancy; OncoSec: Consultancy, Research Funding; PrimeVax: Consultancy, Equity Ownership; Peregrine: Consultancy; UbiVac: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and managing Member of LLC; Ventana/Roche: Consultancy; Viralytics: Consultancy, Research Funding. Bonvini: MacroGenics, Inc.: Employment, Equity Ownership, Research Funding. Wigginton: MacroGenics: Employment, Equity Ownership. Davidson-Moncada: MacroGenics: Employment, Equity Ownership.

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