scholarly journals Recurrent Mutations in CCND3 Confer Clinical Resistance to FLT3 Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 677-677 ◽  
Author(s):  
Catherine C. Smith ◽  
Aaron D. Viny ◽  
Evan S. Massi ◽  
Cyriac Kandoth ◽  
Nicholas D. Socci ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Acquired Resistance ◽  
Cyclin D3 ◽  
Phase Ii Trials ◽  
Advisory Committees ◽  
Primary Resistance ◽  
Clinical Resistance ◽  
Recurrent Mutations ◽  
Flt3 Inhibitors

Abstract Background. Activating mutations in FLT3 occur in ~30% of adult acute myeloid leukemia (AML) cases, including internal tandem duplication (ITD) mutations (~25%) and point mutations in the tyrosine kinase domain (KD). In recent years, multiple selective and potent FLT3 inhibitors such as quizartinib, PLX3397, crenolanib and ASP2215 have demonstrated encouraging preliminary clinical activity in FLT3 mutant AML. Both quizartinib (Schiller et al, ASCO 2014) and ASP2215 (Levis et al, ASCO 2015) have reported composite complete remission rates (CRc) as high as ~50% in phase II trials. Despite these high response rates, the majority of patients relapse after initial response (acquired resistance) and a significant proportion of patients also fail to respond at all (primary resistance). On-target resistance due to secondary KD mutations in FLT3 is a common cause of acquired resistance to the clinically active FLT3 inhibitors quizartinib (Smith et al. Nature 2012) and PLX3397 (Smith et al, Cancer Discovery 2015). However, the causes of primary clinical resistance to FLT3 inhibitors have not been characterized. We performed targeted capture based sequencing of sorted pre-treatment blasts from 8 responding (R) and 21 non-responding (NR) patients treated on the phase I/II trial of PLX3397 in FLT3-ITD+ AML. We deeply sequenced the coding regions of 585 genes known to be mutated in hematologic malignancies/solid tumors and identified genes mutated only in NR patients (compared to genes mutated in both R and NR patients). Results. The number of mutations detected in genes other than FLT3 ranged from 2-18 per sample. There was no increase in the number of mutations found in patients with pre-existing hematologic conditions or in NR patients. Surprisingly, one of the most frequently mutated genes observed exclusively in NR patients was CCND3, the gene encoding cyclin D3, which has rarely been reported to be mutated in AML, though it is mutated in 38% of sporadic Burkitt's lymphoma (BL). A total of 4 individual mutations in CCND3 (Q276*, Q280fs, R271fs, and T283A) were identified in 3/21 NR patients (one patient had both Q276* and Q280fs). No CCND3 mutations were found in R patients. The identified mutations were the same mutations commonly found in BL, known to result in a more stable isoform of cyclin D3 and retain sensitivity to CDK4/6 inhibitors (Schmitz et al., Nature 2012). Expression of the Q276* and T283A mutations in FLT3-ITD+ MV4;11 cells conferred resistance to apoptosis induced by several FLT3 inhibitors (PLX3397, AC220 and crenolanib). However, inhibition of CDK4/6 activity in CCND3 mutant MV4;11 cells by either the CDK4/6 inhibitor palbociclib or the combined FLT3-CDK4/6 inhibitor AMG925 (FLX925) was unable to restore sensitivity to FLT3 inhibition. Moreover, CCND3 mutant MV4;11 cells demonstrated no increase in Rb phosphorylation, suggesting resistance to FLT3 inhibitors facilitated by CCND3 mutations is not predicated on CDK4/6 activation of Rb-dependent E2F-mediated transcription. Conclusions. We have identified recurrent mutations in CCND3, a gene not previously known to be commonly mutated in AML, as a novel cause of clinical primary resistance to FLT3 inhibitors in AML. This represents the first report of a specific non-FLT3 dependent mechanism of clinical resistance to FLT3 inhibitors. Unlike in BL, the functional effects of CCND3 mutations in FLT3-ITD+ AML do not appear to be mediated via CDK4/6 activity and is therefore unresponsive to CDK4/6 inhibition. The molecular mechanism(s) of CCND3-mediated resistance to FLT3 inhibitors are currently being investigated. Disclosures Smith: Plexxikon: Research Funding; Astellas: Research Funding. Off Label Use: Investigational use of PLX3397 in AML. Hsu:Plexxikon Inc.: Employment. West:Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Shah:Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Plexxikon Inc.: Research Funding.

Clinical Resistance To Ruxolitinib Is More Frequent In Patients Without MPN-Associated Mutations and Is Rarely Due To Mutations In The JAK2 Kinase Drug-Binding Domain

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1591-1591 ◽  
Author(s):  
Annalisa Andreoli ◽  
Emmanuelle Verger ◽  
Marie Robin ◽  
Emmanuel Raffoux ◽  
Jean-Marc Zini ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Board Of Directors ◽  
Research Funding ◽  
Kinase Domain ◽  
Drug Binding ◽  
Spleen Size ◽  
Advisory Committees ◽  
Primary Resistance ◽  
Secondary Resistance ◽  
Clinical Resistance

Abstract Background Ruxolitinib (RUX) has been recently approved for the therapy of patients (pts) with myelofibrosis (MF). Although various definitions for response and resistance to this drug have been proposed in the setting of clinical trials, none of them has been validated in clinical practice. Based on RUX efficacy, it is assumed that primary resistance is related to absence or minor reduction in spleen size and constitutional symptoms, while spleen regrowth and recurrence of symptoms after a period with good response establish secondary resistance. The extents of all these parameters have not been clearly defined yet for patients’ management in clinical practice. On the other hand, several biological mechanisms of resistance have been described. In particular, acquisition of new mutations in the predicted RUX-binding region (like Y931C, G935R, R938L, I960V and E985K) was previously shown to confer resistance to JAK inhibitors in vitro (Hornakova et al, Haematologica, 2011; Weigert et al, J Exp Med, 2012). Such mutations have not yet been found in pts treated with RUX. Aims 1) Characterize the parameters leading physicians to conclude that RUX-resistance (RUX-R) was reached in a cohort of consecutive MF pts treated with RUX in clinical practice. 2) Identify a molecular signature of RUX-R. Methods Consecutive pts with MF treated with RUX in our center were identified. Clinical characteristics and evolution during and after RUX therapy were collected. Pts were screened for JAK2V617F (PCR), MPL, TET2, and SRSF2 mutations (direct Sanger sequencing). In addition, the last exons of JAK2 corresponding to the RUX-binding domain were sequenced in RUX-R pts. Results In all, 41 pts received RUX between Nov 2009 and May 2013, including 21 primary, 8 post-PV and 12 post-ET MF, and full data were available in 39 of them for this analysis. Median age was 64 yrs; IPSS risk score was high in 46%, and int-1 or 2 in 54%; 72% were JAK2V617F positive, 10% MPL515-positive. Overall, 16/39 (41%) pts were considered RUX-R by their physician, only 4/16 being primary resistant with <10% reduction in spleen size after a median RUX exposure of 89 days. Median spleen size reduction (best response by palpation, compared with baseline) was 60% in the whole cohort, 50% in pts who developed secondary resistance, and 80% in non RUX-R pts. Secondary resistance was always due to regrowth of spleen either alone (n=6), or associated with recurrence of symptoms (n=5), or with marked leukocytosis (n=1). 44% of RUX-R pts also had hematological intolerance (35% in non RUX-R pts). Median RUX exposure was 298 days (Q1-Q3: 159-640), longer in RUX-R pts compared to non RUX-R pts (median of 383 vs. 292 days). Median starting dose was similar in both groups (15 mg BID), but a higher proportion of pts in the RUX-R group had to reduce the dose < 10 mg BID during follow-up (29% vs 17% in non RUX-R pts). However, 43% of RUX-R was noticed in pts on stable doses, and in 36% after dose escalation (up to 25 mg BID). Among RUX-R pts, there was a higher proportion of pts with high IPSS (56% vs. 39% in non RUX-R; p=0.06), and of post-ET MF (38% vs. 26%; p=0.08). Molecular profile of pts developing RUX-R showed that 31% of them had no mutation detectable at diagnosis in JAK2, MPL, TET2, and SRSF2, compared to 9% of pts in the non RUX-R group (p=0.003). Sequencing of the JAK2 kinase domain in samples taken at the time of resistance in 14/16 RUX-R pts did not detect any new mutation potentially affecting drug binding. Conclusion In this series of consecutive pts treated with RUX in a single center, RUX-R was observed in 41% of pts, mostly as a late event (after median exposure of 1 year) and rarely as primary resistance (10%). RUX-R was always associated with spleen regrowth, and accompanied with recurrence of symptoms in half of the cases. RUX-R was more frequently seen in pts with high risk IPSS score, post-ET MF, smaller spleen response (-50% vs. -80% in non RUX-R), but the only factor significantly associated with RUX-R was the absence of mutation in all of the following genes: JAK2, MPL, TET2, and SRSF2. Furthermore, sequencing of the kinase domain of JAK2 in RUX-R pts didn’t detect any mutation possibly affecting drug binding, suggesting that such mechanism is rarely involved in clinical resistance to RUX. Other mechanisms, like the phenomenon of “persistence” to JAK inhibitor therapy described by Koppikar at al (Nature, 2012) should be investigated in these pts. Disclosures: Robin: Novartis: Research Funding. Rea:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Teva: Honoraria; Ariad: Honoraria. Giraudier:NOvartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria. Kiladjian:AOP Orphan: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Research Funding; Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

A Multicenter, Phase IV Observational Study Of Ofatumumab In Chronic Lymphocytic Leukemia (CLL): A European Research Initiative On CLL (ERIC) Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1645-1645 ◽  
Author(s):  
Carol Moreno ◽  
Marco Montillo ◽  
Panayiotidis Panayiotis ◽  
Adrian Bloor ◽  
Jehan Dupuis ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Adverse Events ◽  
Board Of Directors ◽  
Safety Profile ◽  
Research Funding ◽  
Response Rate ◽  
Lymphocytic Leukemia ◽  
Hematologic Toxicity ◽  
Phase Ii Trials ◽  
Advisory Committees

Abstract Background Ofatumumab was given a conditional approval in the EU on April 2010 for the treatment of CLL refractory to fludarabine (F-ref) and alemtuzumab (A-ref), encouraging the retrieval of further data in patients treated in a “daily life” setting and to investigate treatment safety. Aims The main objective of this study was to obtain information on the safety profile of ofatumumab given outside clinical trials in patients with previously treated CLL. The secondary endpoints were efficacy, progression-free-survival (PFS), and overall survival (OS). Methods This was an observational, retrospective study. Patients were eligible regardless of prior treatments or disease status and provided they had not been included in ofatumumab clinical trials. Data on patients’ characteristics at diagnosis, prior treatment, adverse events response rate, PFS and OS were recorded. Results One-hundred and twenty patients were screened of which 103 from 25 centers in 10 European countries were eventually eligible for the study. There were 71 males; median age at initiation of ofatumumab was 64 years (range, 38-84); 66% patients were in advanced clinical stage (Rai III-IV/Binet C) and 33 patients presented bulky lymphadenopathy. Number of prior lines of therapy was 4 (range, 1-13). 94% had received prior F-based therapy, 54% received A-based therapy and 51% received both. Eighty-one percent had been previously exposed to rituximab-combination regimens. Fifty-four percent were F-ref, 70% A-ref and 41% were both F- and A-refractory. Cytogenetics within 3 months prior therapy was available in 52 patients of which 34 presented abnormalities (11 patients: 17p-; 9 patients: 11q-; 2 patients: 13q-; 1 patient: trisomy 12; 11 patients: two or more abnormalities including 17p- or 11q-). Forty-two of 50 patients showed unmutated IGHV genes. The median number of cycles of ofatumumab given was 9 (range, 0-16) and the median percentage of given/planned cycles was 83.3% (range, 0-133). In most patients the treatment dose and schedule were as follows: 300 mg 1st infusion followed by 2000 mg for subsequent infusions (8 weekly followed by 4 doses monthly). One hundred and sixty-one adverse events were reported in 68 patients, with 28 (17%) of them being considered as ofatumumab-related. Infusion related-reactions occurred in 19 (28%) patients (III-IV: 6%). Neutropenia was reported in 26% (III-IV: 19%), thrombocytopenia in 15% (III-IV: 12%) and anemia in 15% (III-IV: 7%). The non-hematological adverse events, included infection 44% (III-IV: 36%), fatigue 10% (III-IV: 4%), fever 10% (III-IV: 6%), rash 10% (III-IV: 3%), cough 7% (III-IV: 1%), diarrhea 6% (grade III-IV: 0%) and nausea 1% (III-IV: 0%). Hematologic toxicity correlated with the number of prior lines of therapy. Autoimmune hemolytic anemia and Richter syndrome were reported in one patient each. Two heavily pre-treated patients (5 and 6 prior lines of therapy, respectively) developed progressive multifocal leukoencephalopathy. The overall response rate (ORR) was 23% and the median PFS and OS were 5 and 12 months, respectively. The main causes of death were disease progression (61%) and infection (28%). Conclusions The safety profile of ofatumumab given outside clinical trials to patients with poor-prognosis and heavily pre-treated CLL was consistent with that observed in clinical trials. Although not unexpectedly the ORR was lower in this study, PFS and OS were in line with those reported in phase II trials. Disclosures: Montillo: Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Bloor:GSK: Consultancy, Honoraria, Paid speaker Other. Schuh:GSK: Honoraria; Celgene: Honoraria; Mundipharma: Honoraria. Geisler:Roche: Consultancy; GSK: Consultancy. Hillmen:GlaxoSmithKline: Honoraria, Research Funding. Stilgenbauer:GSK: Honoraria, support Other. Smolej:GSK: Consultancy, Honoraria, travel grants Other. Jaeger:GSK: Honoraria, Research Funding. Leblond:Roche : Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Mundipharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kimby:Roche: Consultancy, Honoraria, Research Funding; celgene: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding; Emergent BioSolutions: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding; Jansen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding.

Outcome of Lower Risk Non Del 5q MDS after Failure of Erythropoiesis Stimulating Agents (ESA), and Impact of Post-ESA Treatment on Survival: A Retrospective European Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1665-1665 ◽  
Author(s):  
Sophie Park ◽  
Jean-François Hamel ◽  
Andrea Toma ◽  
Charikleia Kelaidi ◽  
Maria Campelo Diez ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Lower Risk ◽  
Research Funding ◽  
Cooperative Groups ◽  
Second Line ◽  
Advisory Committees ◽  
Primary Resistance ◽  
Investigational Drugs ◽  
Significant Difference ◽  
Very High

Abstract Background : Most non-del 5q lower risk MDS patients (pts) are first treated with ESA, with about 50% (generally transient) responses, and second line treatments (TX) including hypomethylating agent (HMA), Lenalidomide (LEN) and investigational drugs are then often proposed, but their effect on overall survival (OS) is unknown. In a previous work on 253 such pts, we found worse OS with early failure to ESA, i.e. primary resistance (RES) or relapse (REL) < 6 months after ESA onset (Kelaidi, Leukemia, 2013), but only few pts had received, after ESA failure, TX other than RBC transfusions. In the present study, we gathered non-del 5q lower risk MDS treated with ESA from several EU MDS cooperative groups, and analyzed their outcome after ESA failure, and the effect of second line TX on survival. Methods : 1611 IPSS low and int-1 (lower risk) non del 5q MDS pts included in the French (GFM), Italian (FISM), Spanish (GESMD), Greek, Düsseldorf and Munich registries between 1997 and 2014, and treated by ESA were studied. Survival was assessed from failure of ESA (i.e. from primary failure evaluated after 12 to 24 weeks of ESA treatment, or from relapse after a response). Progression at ESA failure was defined upon progression to a higher IPSS-R class at ESA failure as compared with ESA onset. Results : At ESA onset, the 1611 pts were reclassified by IPSS-R in 16% very low, 54% low, 13% int, 6% high, 1% very high and 10% ND. HI-E (using IWG 2006 criteria) to ESA treatment was 66.9%, and the median duration of response was 15 months. The cohort of 1038 pts with ESA failure included 521 RES and 517 REL. Median OS was 4.2 years in REL and 3.7 years in RES pts (p=0.56), and no significant difference was seen, even after restricting the analysis to very low and low IPSS-R pts (p=0.81), or when analyzing "early" vs "late" failures, with cut-off points at 6 or 12 months, as we previously reported (Kelaidi, Leukemia, 2013). 336 (32%) pts received second line treatment (TX2) other than RBC transfusions, including HMA in 88 pts, LEN in 169 pts, and other TX (OT) in 79 pts (including 11 chemotherapy, 17 thalidomide, 11 immunosuppressors (ATG, cyclosporine), or investigational drugs), with response rates of 46%, 39% and 33% respectively (p=0.4). 87 pts had a third line TX (mostly a new drug, but also 7 pts who received HMA after LEN, and 33 pts LEN after HMA). Pts treated with LEN as TX2 were younger (median age 70 vs 75 for BSC, and 70 for HMA p<10-4), had more RARS (67% vs 28% for BSC and 27% for HMA, p<10-4), while pts treated with HMA as TX2 had more RAEB-1 (34% vs 10% for BSC and 12% for LEN, p<10-4) and more high and very high IPSS-R at onset of TX2 (48% vs 4.6% for BSC and 3.1% for LEN, p<10-4). Median OS for pts receiving BSC, LEN, HMA and OT as TX 2 was 4.3y, 3.7y (HR 1.1 [0.81-1.50] p=0.5), 2.1y (HR 1.59 [1.12-2.72], p=0.01) and 2.2y (HR1.17 [0.81-1.68], p=0.41) respectively (Figure). However, in a multivariate analysis adjusted on age, gender, and IPSS-R progression at ESA failure, OS difference became not significant. Analysis of AML progression in the different TX2 groups is currently being finalized. C onclusion: In this large multicenter retrospective cohort of non-del 5q lower risk MDS pts having failed ESA treatment, OS from failure was similar in RES and REL pts, contrary to our previous smaller experience. About 1/3 of the pts received second line treatments other than RBC transfusion, mainly LEN or HMA. However, none of those treatments was able to improve OS compared to BSC. Newer treatments are required in this situation, possibly including allogeneic SCT in younger pts. Figure 1. OS since ESA failure according to TX2 (Simon-Makuch method). Figure 1. OS since ESA failure according to TX2 (Simon-Makuch method). Disclosures Park: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding; Celgene: Research Funding. Off Label Use: Lenalidomide in non del 5q MDS. Santini:celgene, Janssen, Novartis, Onconova: Honoraria, Research Funding. Cony-Makhoul:BMS: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Cheze:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wattel:PIERRE FABRE MEDICAMENTS: Research Funding; CELGENE: Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding; NOVARTIS: Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding. Vey:Celgene: Honoraria; Roche: Honoraria; Janssen: Honoraria. Fenaux:Amgen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

scholarly journals A Phase-Ib/II Clinical Evaluation of Ponatinib in Combination with Azacitidine in FLT3-ITD and CBL-Mutant Acute Myeloid Leukemia (PON-AZA study)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2350-2350
Author(s):  
David Kipp ◽  
Sun Loo ◽  
Andrew Charles Perkins ◽  
Steven W Lane ◽  
Emily Blyth ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Loss Of Function ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Best Response ◽  
Flt3 Inhibitor ◽  
Flt3 Inhibitors ◽  
Eliza Hall Institute ◽  
Hall Institute

Abstract Introduction Despite the advent of targeted therapy for FLT3-mutated AML, unmet need still exists for patients unfit for intensive chemotherapy, with no evidence that overall survival (OS) can be improved by combining either venetoclax (Konopleva et al., ASH 2020) or gilteritinib (Astellas press release, December 2020) with azacitidine. Although gilteritinib has been shown to improve median OS from 5.5 to 9.8 months, the majority will relapse (Perl et al., 2019). Adaptive on-target gilteritinib resistance may be due to the FLT3-F691L gatekeeper mutation, whereas off-target resistance may be due to loss-of-function variants in CBL, which encodes an E3 ubiquitin-protein ligase that negatively regulates FLT3 (McMahon et al, 2019). Ponatinib is a type-1 FLT3 inhibitor that is active in vitro against FLT3 F691L (Smith et al., 2013) and had an overall response rate (ORR) of 43% in a small pilot phase-I study (Talpaz et al., 2011). Combination of a FLT3 inhibitor with azacitidine may antagonize the synergistic hypermethylation reported for FLT3-ITD in association with epigenetic mutations (Shih et al., 2015). CBL loss-of-function mutations may also enhance responsiveness to FLT3 inhibitors (Taylor et al, 2015). We thus hypothesize that the combination of ponatinib and azacitidine could mitigate the rapid evolution of drug resistance typical of more selective FLT3 inhibitors used as single agents. Methods A phase-Ib study was conducted with the primary objective safety and key secondary objective preliminary efficacy of azacitidine in combination with ponatinib in patients with FLT3-ITD AML failing prior therapy or unfit for intensive chemotherapy. Exploratory objectives included mechanisms of ponatinib resistance and responsiveness of CBL-mutant AML to FLT3 inhibition. At dose level 1 (DL1), patients received azacitidine 60 mg/m 2 on days 1-5 and 8-9 and ponatinib 30 mg daily on days 5-25 of each cycle. In patients not achieving CR or CRi after cycle 1, the ponatinib dose was increased to 45 mg during cycle 2. For dose level 2 (DL2), the dose of azacitidine was increased to 75 mg/m 2. Results Thirty-one patients were evaluable for response. Median age was 67 years (range, 26-87). Frequency of prior lines of therapy was 0 (15%), 1 (46%), 2 (23%) or 3 (8%). Four patients had a history of prior allogeneic hematopoietic cell transplant and one had previously received a FLT3 inhibitor. FLT3-ITD was present in 28 patients (median VAF 0.33; range, 0.009-17.95) and 3 had inactivating CBL mutations. A total of 20 patients were treated at DL1 and 12 patients at DL2. There were two grade-4 DLTs (raised AST/ALT [DL1] and tubulointerstitial nephritis [DL2]). Three grade-2 thromboembolic events were observed (two cannula-related DVTs and a distal lower-limb DVT). There were two grade-5 AEs (infection and cardiac failure), which were not considered drug related. The most common grade-3-4 AEs were febrile neutropenia (57%), neutropenia (47%), infections (47%), thrombocytopenia (40%) and anaemia (27%). Cardiac arrhythmias (atrial fibrillation/flutter, bradycardia, sinus tachycardia and ventricular tachycardia [1 patient]) were observed in 30% of patients. Of these, 80% were grade 1 or 2 and only one was considered by the investigator to be related to study treatment. Response was evaluable in 23 of 31 patients. Nine patients (39%) achieved CR or CRi, 3 (13%) achieved a PR and 8 (35%) achieved SD (ORR 52%). ORR at DL1 and DL2 was 43% and 66%, respectively. Median time to best response was 1.4 months (range 1.0-11.9). Median duration of best response was 12.9 months at both dose levels. Median OS for DL1 was 6.5 months and not reached for DL2. Despite shorter follow-up, DL2 patients experienced better OS than DL1 patients (p = 0.015). Responses were seen in 2 of 4 patients with post-allograft relapse. Two of three patients with a CBL mutation responded (1 CR and 1 CRi). Eradication of the CBL mutation was seen in one patient, who remains on therapy after 15 cycles. Molecular studies to investigate dynamic changes in molecular architecture are ongoing. Conclusions The recommended phase-II dose of ponatinib is 30 mg on days 5-25 and that of azacitidine is 75 mg/m 2 for seven doses each cycle. The ORR was 52% and durable disease control was observed, especially in patients receiving DL2. Preliminary efficacy was observed in CBL-mutated patients. Further clinical investigation of this regimen is warranted in patients with FLT3- or CBL-mutant AML. Figure 1 Figure 1. Disclosures Kipp: Novartis: Honoraria. Perkins: Celgene: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau. Lane: Novartis: Consultancy; Geron: Consultancy; BMS: Consultancy, Research Funding; Abbvie: Honoraria; Astellas: Membership on an entity's Board of Directors or advisory committees. Enjeti: Sanofi: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria; Roche: Speakers Bureau; Astra Zeneca: Honoraria. Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Reynolds: Novartis AG: Current equity holder in publicly-traded company; Abbvie: Research Funding; Alcon: Current equity holder in publicly-traded company. Wei: Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Membership on an entity's Board of Directors or advisory committees; Novartis, Abbvie, Celgene/BMS: Speakers Bureau; Former employee of Walter and Eliza Hall Institute: Patents & Royalties: Prof. Andrew Wei is a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax; Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Honoraria; Servier: Consultancy; Abbvie, Amgen, AstraZeneca, Celgene/BMS, Novartis, Servier and F. Hoffmann-La Roche: Research Funding. OffLabel Disclosure: Ponatinib - used as an experimental therapy for AML in combination with azacitidine

scholarly journals The FLT3 F691L Gatekeeper Mutation Promotes Clinical Resistance to Gilteritinib + Venetoclax (GILT + VEN) in AML

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2235-2235
Author(s):  
Setareh Sharzehi ◽  
Sunil K Joshi ◽  
Janét Pittsenbarger ◽  
Jeffrey W. Tyner ◽  
Elie Traer
Keyword(s):  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Research Funding ◽  
Immunoblot Analysis ◽  
Tyrosine Kinase Domain ◽  
Resistance Mutations ◽  
Advisory Committees ◽  
Mechanism Of Resistance ◽  
Clinical Resistance

Abstract Background: FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown. Results: Early resistance cell cultures to GILT+VEN were created by exposing MOLM14 cells to GILT 25nM + VEN 25nM alone or supplemented with microenvironmental ligands FGF2 or FLT3 ligand (FL; N=3/group). Media, drugs, and ligands were replenished twice weekly. After 25 weeks, only the cultures exposed to ligand resumed growth (N=1 for FGF2 and N=3 for FL). Ligands were then removed from these early resistant cultures to induce late resistance. There was an initial drop in cell viability but cells resumed growth after only 3.5 weeks (Fig. 1). In contrast, the time to develop early and late resistance to GILT monotherapy was 8 and 15 weeks, respectively. Immunoblot analysis of GILT + VEN early and late resistant cultures demonstrated restoration of FLT3 signaling, as well as phosphorylation of downstream AKT/MAPK pathways. These results also contrasted to late GILT monotherapy resistant cultures, which had downstream AKT/MAPK activation via outgrowth of NRAS mutations. Since FLT3 appeared to be functionally active, we sequenced FLT3 and found that all early and late GILT + VEN resistance cultures had gatekeeper FLT3 F691L mutations. F691L accounted for only in a minority of resistance cultures to GILT monotherapy. To test if FLT3 signaling was important for resistance, we exposed parental cells to higher concentrations of gilteritinib, which have been shown to partly overcome F691L, as well as the FLT3i FF-10101, which binds FLT3 at a different site and is not affected by the F691L mutation. Both of these approaches restored sensitivity to FLT3i in vitro. As expected, the F691L mutation provided broad resistance to most FLT3i (Fig. 2). To validate this mechanism of resistance in patients, we identified a relapsed FLT3-ITD patient who was treated with GILT monotherapy for 5 months, followed by GILT + HMA for 4 cycles, and then GILT + VEN for resistant proliferative disease. After an initial response to GILT + VEN, the leukemia cells began to increase again in the peripheral blood. A repeat genetic test was ordered and the patient was found to have developed a FLT3 F691L mutation at a high variant allele frequency (Fig. 3). Conclusion: We have developed a robust cell line model of early and late resistance to FLT3i that mimics the timing and expansion of resistance mutations in the clinic. Our model of early and late resistance to GILT combinations can prospectively predict mechanisms of resistance. Although uncommon as a mechanism of resistance to GILT monotherapy, our model and early patient data predicts that F691L mutations are more important for GILT + VEN resistance. Figure 1 Figure 1. Disclosures Tyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Marcks Marks Resistance to Proteasome Inhibitors: Exocytosis of Polyubiquitinated Proteins in Bortezomib-Resistant Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3712-3712
Author(s):  
Niels Franke ◽  
Gertjan J.L. Kaspers ◽  
Yehuda G Assaraf ◽  
Johan Van Meerloo ◽  
Denise Niewerth ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Acquired Resistance ◽  
Proteasome Inhibitors ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Resistance Level ◽  
Proteolytic Stress ◽  
Resistant Cells

Abstract Despite the proven efficacy of proteasome inhibitors like bortezomib (BTZ) in multiple myeloma, mantle cell lymphoma, and in an experimental setting in pediatric acute leukemia, development of drug resistance remains a primary hindrance. To further understand the molecular basis underlying this chemoresistance phenomenon, various leukemia cell line models with acquired resistance to BTZ were developed and characterized. One common characteristic was that acquisition of point mutations in PSMB5 and upregulation of the β5-subunit of the proteasome were key determinants of BTZ-resistance in vitro. However, it remains unclear how these drug resistance modalities translate to the overcoming of proteolytic stress imposed by proteasome inhibition. From this perspective, we here undertook a multi-modality (DNA, mRNA, miRNA) array-based analyses of human CCRF-CEM acute leukemia cells and two BTZ-resistant subclones [one with a low resistance level [(10-fold, CEM/BTZ7) and another subline with a high resistance level (140-fold, CEM/BTZ200)] to determine whether or not complementary mechanisms contribute to BTZ resistance. Gene expression profiling studies revealed markedly reduced proteolytic stress induction in drug resistant cells over a broad BTZ concentration range. Moreover, several genes involved in cytoskeleton regulation and vesicle migration were increased in resistant cells. Of all genes, myristoylated alanine-rich C-kinase substrate (MARCKS) was the most differentially overexpressed gene with 25- to 42-fold upregulation in CEM/BTZ7 and CEM/BTZ200 cells, respectively. These observations were corroborated at the protein level and solely included unphosphorylated MARCKS rather than phosphorylated MARCKS, which was marginally expressed in CEM/BTZ cells. Interestingly, MARCKS upregulation was also observed in other BTZ-resistant and leukemia cells (CEM and THP1) with acquired resistance to the proteasome inhibitor salinosporamide A and the immunoproteasome inhibitor PR924. Given the overexpression of MARCKS in proteasome inhibitor-resistant leukemia cells, we further explored whether or not MARCKS overexpression may serve as a predictive marker of BTZ resistance in clinical samples of acute leukemia patients. To this end, we examined primary patient specimens from a phase II childhood refractory/relapsed ALL trial in which BTZ is administered in two intensive re-induction regimens containing vincristine, prednisone, PEG-asparaginase, doxorubicin or cyclophosphamide and etoposide followed by methotrexate treatment. MARCKS expression was demonstrated in 64% of therapy-refractory pediatric leukemia specimens (n=44) wherein higher MARCKS expression trended (p=0.09) towards a dismal response to BTZ-containing chemotherapy. Finally, from a mechanistic perspective, we showed a concentration-dependent association of MARCKS protein with the emergence of ubiquitin-containing vesicles in the resistant cells. This association with MARCKS protein was lost upon exocytosis of these vesicles, which were found to be extruded and taken up in co-cultures with recipient HeLa cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance through vesicular exocytosis of ubiquitinated proteins in BTZ-resistant cells to overcome proteolytic stress over a broad range of cytotoxic BTZ concentrations. Disclosures Kaspers: Janssen-Cilag: Research Funding. Smeets:Novartis: Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cloos:Takeda: Honoraria.

scholarly journals Mutations in Driver Genes and Changes in Clonal Dynamics Are Associated with Shorter Time to Treatment in MBL Cases

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5264-5264
Author(s):  
Santiago Barrio ◽  
Juhi Ojha ◽  
Charla Secreto ◽  
Kari G. Chaffee ◽  
klaus Martin Kortum ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Germ Line ◽  
Clonal Evolution ◽  
Clonal Expansion ◽  
Driver Mutations ◽  
Driver Gene ◽  
Driver Genes ◽  
Advisory Committees ◽  
Recurrent Mutations

Abstract Introduction: Monoclonal B cell lymphocytosis (MBL) is an asymptomatic expansion of clonal CD19+/CD5+ B cells with less than 5x109/L cells in the peripheral blood and without other manifestations of chronic lymphocytic leukemia (CLL). Approximately 1% of MBL evolves to CLL requiring therapy per year; thus it is critical to develop more precise tools to identify which MBL will progress to CLL and require treatment. Patients and Methods: In this study, we performed targeted deep sequencing (TDS) on 49 high-count MBL individuals (median B-cell count 3.7x109/L; range 0.8-4.9x109/L) and explored the mutation status of 20 driver genes recurrently mutated in CLL. We analyzed the clonal evolution in 45 of these 49 MBLs by screening 2-4 sequential samples (average time between samples 56 months, range 10-119 months). At last follow-up, 19 cases (39%) had progressed to Rai>0, and 10 cases (20%) required treatment. Tumor and germ line DNAs were isolated from sorted CD5+/CD19+ and CD5-/CD19- cell populations, respectively. Overall, 154 samples from 49 MBL cases (105 tumor and 49 germ line) were screened using semiconductor sequencing technology. The latter genetic information was integrated with relevant clinical and biological parameters, and we evaluated the effect of driver mutations and clonal expansion on time to CLL progression and time to treatment (TTT). Results and Discussion: Our cohort consisted in 17 women and 32 men, with a median age of 66 years (range: 44-80). Five cases presented secondary diseases, including melanoma, lung and bladder cancer. Clinical and biological parameters were collected, including IGHV mutation status (mutated 66%, unmutated 34%), ZAP70 and CD49 expression (25% each). At presentation, 46% of cases had del(13q), 27% trisomy 12, 6% del(11q), and 4% del(17p). Overall, we found somatic non-synonymous mutations in 23 of 49 MBLs (47%) at the initial time point including 22% of cases with more than one mutated driver gene. The average depth of coverage was 730x, thus allowing the identification of small subclonal mutations. Recurrent mutations were found in most of the drivers: CHD2, DDX3X (8% of cases), FBXW7, NOTCH1, SF3B1 (6% each), ATM, BCOR, BIRC3, BRAF, KRAS, MED12, MYD88 and ZMYM3 (4% each). Furthermore, ITPKB, POT1, SAMHD1 and XPO1 were mutated in only one case, whereas no mutations were found in HIST1H1E, RIPK1 and TP53. In 4 individuals, we found two mutations in the same gene (BRAF, DDX3X, KRAS and SAMHD1). Genes that are known to be associated with disease progression in CLL were either mutated with significantly lower incidence (NOTCH1, SF3B1) or not mutated (TP53). Mutations were detected on average 45 months (range 9-73) prior to progression to CLL Rai>0 indicating the early origin of most driver gene mutations in the MBL/CLL continuum. The presence of driver mutations in MBL was associated with shorter TTT (median TTT: present: 96 months vs. not present: not reached, HR: 5.52, 95% CI: 1.2-26.2, P =0.015). Next, we looked at clonal expansion of driver mutations over time (defined as >2-fold change in the allelic frequency of driver mutations between time points). Of 20 MBLs with mutations at baseline who had sequential samples available, 10 cases showed clonal expansion. Seven out of 10 MBLs who required therapy showed clonal expansion, which was detected on average 15 months (range 6-30 month) prior to treatment. Finally, the detection of clonal expansion was significantly associated with reduced TTT (median TTT: clonal expansion: 21 months vs. no clonal expansion: 84 months, HR: 7.79, 95% CI: 1.94-31.3, P <0.001). Conclusion: We have confirmed the existence of recurrent mutations in most CLL putative driver genes at the premalignant MBL stage many years before progression to CLL. Furthermore, the early identification of driver mutations and its clonal expansion predicts a shorter TTT. Of note, clonal evolution under selective pressure has recently been linked to the onset of CLL progression after therapy. In this study, we characterized the clonal dynamics in the pre-malignant stages of the disease and underlined its impact on clinical outcome. Despite the relatively small size of the cohort, these findings suggest that the sequential monitoring of MBL individuals with a simple and reliable technique, such as TDS, will be at least of prognostic use and thus its incorporation in the disease stratification and clinical management should be further tested. Disclosures Fonseca: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Applied Biosciences: Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Binding Site: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Onyx/Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Tolero Pharma: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Hospira: Research Funding.

scholarly journals Overcoming Adaptive Therapy Resistance in AML By Targeting Immune Response Pathways

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3934-3934
Author(s):  
Katelyn M. Melgar ◽  
LaQuita M Jones ◽  
Mackenzie Walker ◽  
Lyndsey C Bolanos ◽  
Kathleen Hueneman ◽  
...  
Keyword(s):  
Stress Response ◽  
Tyrosine Kinase ◽  
Board Of Directors ◽  
Small Molecule ◽  
Research Funding ◽  
Response To Therapy ◽  
Innate Immune ◽  
Advisory Committees ◽  
Adaptive Resistance ◽  
Flt3 Inhibitors

Targeted inhibitors to oncogenic kinases demonstrate encouraging clinical responses early in the treatment course, however, most patients will relapse due to target-dependent mechanisms that mitigate enzyme-inhibitor binding or through target-independent mechanisms, such as alternate activation of survival and proliferation pathways, known as adaptive resistance. One example involves the FMS-like receptor tyrosine kinase (FLT3). Activating mutations of FLT3 result in its autophosphorylation and initiation of intracellular signaling pathways, which induce abnormal survival and proliferation of leukemic cells.One of the most common mutations in acute myeloid leukemia (AML) involves the internal tandem duplication (ITD) of FLT3, which occurs in ~25% of all cases of newly diagnosed AML and confers a particularly poor prognosis. FLT3 inhibitors (FLT3i) evaluated in clinical studies as monotherapy and combination therapies have shown good initial response rates; however, patients eventually relapse with FLT3i-resistant disease. The absence of durable remission in patients treated with potent and selective FLT3i highlights the need to identify resistance mechanisms and develop additional treatment strategies. Several mechanisms contribute to resistance to selective FLT3i, including mutations in the tyrosine kinase domain of FLT3 (20-50%) or activation of parallel signaling mechanisms that bypass FLT3 signaling, referred to as adaptive resistance (30-50%). Here we describe mechanisms of adaptive resistance in FLT3-mutant AML by examining in-cell kinase and gene regulatory network responses after oncogenic signaling blockade by FLT3 inhibitors (FLT3i). Through this integrative approach, we identified activation of innate immune stress response pathways after treatment of FLT3-mutant AML cells with FLT3i. Utilizing genetic approaches, we demonstrated that innate immune pathway activation via IRAK1 and IRAK4 contributes to adaptive resistance in FLT3-mutant AML cells. The immediate nature of IRAK1/4 activation in adaptively resistant FLT3-ITD AML cells requires concomitant inhibition of these targets to avoid compensatory signaling and cell survival. Achieving optimal multi-drug combination regimens that yield extended overlapping exposure while avoiding unwanted toxicities is challenging. Therefore, we desired a small molecule inhibitor that simultaneously targeted the FLT3 and IRAK1/4 kinases to eradicate adaptively resistant FLT3-ITD AML. To overcome this adaptive resistance mechanism, we developed and optimized a novel small molecule that simultaneously inhibits FLT3 and IRAK1/4 kinases. The FLT3-IRAK1/4 inhibitor exhibited potent binding affinity for IRAK1 (KD= 2.9 nM), IRAK4 (KD= 0.3 nM), and FLT3 (KD= 0.3 nM), as well as acceptable pharmacokinetic properties in mice. Moreover, a high-resolution crystal structure demonstrates that the FLT3-IRAK1/4 inhibitor binds as a type I inhibitor (ATP-competitive binding to the active state). The FLT3-IRAK1/4 inhibitor eliminated adaptively resistant FLT3-mutant AML cell lines and patient-derived samples in vitro and in vivo, and displayed superior efficacy as compared to current targeted FLT3 therapies. Our study demonstrates that therapies that simultaneously inhibit FLT3 signaling and compensatory IRAK1/4 activation have the potential to improve the therapeutic efficacy in patients with FLT3-mutant AML. In conclusion, these findings reveal that inflammatory stress response pathways contribute to adaptive resistance in FLT3-mutant AML and suggests that this mechanism may extend to other malignant cells undergoing a stress-induced response to therapy. Disclosures Hoyt: Kurome Therapeutics: Consultancy. Berman:Astellas: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria; Lilly: Honoraria; Gilead: Consultancy; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Roche: Consultancy, Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Loxo: Membership on an entity's Board of Directors or advisory committees. Rosenbaum:Kurome Therapeutics: Consultancy, Employment. Perentesis:Kurome Therapeutics: Consultancy. Starczynowski:Kurome Therapeutics: Consultancy.

scholarly journals Recurrent Mutations in Cyclin D3 Confer Clinical Resistance to FLT3 Inhibitors in Acute Myeloid Leukemia

2021 ◽  
Author(s):  
Catherine C. Smith ◽  
Aaron D. Viny ◽  
Evan Massi ◽  
Cyriac Kandoth ◽  
Nicholas D. Socci ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cyclin D3 ◽  
Clinical Resistance ◽  
Recurrent Mutations ◽  
Flt3 Inhibitors ◽  
Acute Myeloid

scholarly journals ERK1/2 Inhibition Overcomes Resistance in Acute Myeloid Leukemia (AML) and Alters Mitochondrial Dynamics

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3338-3338
Author(s):  
Priyanka Sharma ◽  
Sujan Piya ◽  
Huaxian Ma ◽  
Natalia Baran ◽  
Malgorzata Anna Zal ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Acquired Resistance ◽  
Mapk Signaling ◽  
Advisory Committees ◽  
Resistance To Therapy ◽  
Erk Inhibition ◽  
Dual Mechanism

Abstract Background: Presence at diagnosis or acquisition of activating RAS pathway mutations is a pervasive mechanism of resistance to therapy in AML. Efforts to directly target mutant RAS have been unsuccessful and the efficacy of BRAF and MEK inhibitors has been limited due to compensatory reactivation of MAPK signaling. ERK1/2 (ERK) is a key downstream component in the MAPK pathway and therefore represents an attractive target for inhibiting MAPK signaling. Compound 27 (1) is a dual-mechanism inhibitor of ERK that inhibits both the catalytic activity of ERK and its phosphorylation by MEK. It is a close analog of ASTX029, a dual-mechanism ERK inhibitor currently under clinical investigation in solid tumors (NCT03520075). Objectives: We analysed the preclinical activity of Compound 27 in AML, investigated its mechanism of action and ability to overcome resistance. Results: Using a panel of 9 AML cell lines, the IC50 value for single agent Compound 27 was in the low to intermediate nanomolar range (1.89-388 nM). Decreased ERK phosphorylation was confirmed by Western blot analysis. To better characterize the biological effects of Compound 27, we performed mass cytometry (CyTOF) analysis of NRAS-mutated OCI-AML3 cells. This experiment showed approximately 75% downregulation of CyclinB1 and cMyc in 250 nM drug-treated cells versus untreated cells (Figure 1a). The expression of anti-apoptotic proteins, including MCL1, BclXL and Bcl2, were also decreased. Western blot analysis confirmed increased cleaved PARP, and reduced cMyc and cell cycle-related proteins CyclinB1, CyclinD1 and CDK4 with Compound 27 treatment. In isogenic cells, p53 knock-down had no effect on the efficacy of Compound 27. We next investigated the efficacy of simultaneous inhibition of ERK and Bcl-2 in AML cells. Compound 27 sensitized OCI-AML3 cells, which are intrinsically resistant to ABT-199 (a Bcl-2 inhibitor), to treatment with ABT-199 and shifted the cytostatic effect of the single agents to a cytotoxic effect with a combination index (CI) of 0.008 (cell death 91% for combination versus 20% with ABT-199 alone). This suggests strong synergistic effects of combination treatment (Figure 1b). In OCI-AML2 cells with acquired resistance to ABT-199, the combination increased apoptosis to 80% as compared to 20% with ABT-199 alone. Compound 27 sensitized bulk CD45+ as well as CD34+CD38-leukemia progenitor cells to ABT-199. Compound 27 also sensitized FLT3-ITD mutant human AML cell lines MOLM13, MOLM14, MV-4-11 and murine Ba/F3-ITD cells to the FLT3 inhibitor AC220 (CI in MOLM13=0.3). Synergy of Compound 27 and 5-azacitidine was also observed (p=0.009). Leukemia microenvironment-mediated resistance to therapy is partly mediated by MAPK activation. We co-cultured OCI-AML3 and MOLM13 cells with normal bone marrow-derived mesenchymal stromal cells (NMSCs) to mimic the bone marrow microenvironment and analysed the effect of Compound 27 in combination with either ABT-199 or AC220. Combination drug treatment were more effective in terms of cytoreduction and apoptosis induction in coculture. However, neither combination was able to completely overcome stroma-mediated resistance (Figure 1b). Analysis of other stroma-relevant molecules in coculture showed that CXCR4 was increased while CD44 was decreased in response to ERK inhibition. Effective reactive oxygen species (ROS) mitigation and hyper-active mitochondrial fission is important for maintaining "stemness" of AML cells (2). ERK phosphorylates DRP1, which is necessary for mitochondrial fission. Treatment of OCI-AML3 cells with Compound 27 led to increased mitochondrial ROS, decreased levels of pDRP1(Ser616) and increased mitochondrial length, suggesting impaired fission and reduced "stemness" of AML cells (Figure 1c). Conclusion: ERK inhibition by Compound 27 synergizes with 5-azacitidine, ABT-199 and AC220 and can overcome primary or acquired resistance. The impact on mitochondrial dynamics suggests a potential impact on leukemia stem cells. Additional mechanistic confirmatory work is in progress. References: 1. Heightman TD, Berdini V, Braithwaite H, et al. Fragment-based discovery of a potent, orally bioavailable inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2. J Med Chem. 2018;61(11):4978-4992. 2. Schimmer AD. Mitochondrial Shapeshifting Impacts AML Stemness and Differentiation. Cell Stem Cell. 2018;23(1):3-4. Figure 1 Figure 1. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Dao: Astex Pharmaceuticals, Inc.: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Andreeff: Medicxi: Consultancy; Syndax: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Oxford Biomedica UK: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees. Borthakur: University of Texas MD Anderson Cancer Center: Current Employment; Takeda: Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; ArgenX: Membership on an entity's Board of Directors or advisory committees; Protagonist: Consultancy; GSK: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document