Single-cell DNA sequencing reveals complex mechanisms of resistance to quizartinib

Peretz, Cheryl A.C.; McGary, Lisa H.F.; Kumar, Tanya F; Jackson, Hunter; Jacob, Jose; Durruthy-Durruthy, Robert; Levis, Mark J.; Perl, Alexander E.; Huang, Benjamin; Smith, Catherine C.

doi:10.1182/bloodadvances.2020003398

Cited by 23 publications

(17 citation statements)

References 21 publications

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“…Acquired resistance to FLT3 inhibitors is a major challenge of the current therapeutic paradigm of FLT3 -mutant AML. Single-cell genomic analyses of patients treated with FLT3 inhibitors have illustrated the heterogenous nature of on-target and off-target resistance mechanisms (12–13, 15). Our group and others have demonstrated that under FLT3 inhibition, clones that re-activate the RAS/MAPK pathway account for clinical resistance in a large proportion of patients (13, 15, 59).…”

Section: Discussionmentioning

confidence: 99%

“…Our group and others recently identified the emergence of polyclonal RAS/MAPK pathway mutations, most commonly in the form of activating NRAS mutations, in patients relapsing on FLT3i (13–15). Functional and biochemical analysis of AML cell lines implicate re-activation of MAPK signaling as a primary mediator of acquired resistance to FLT3i (13).…”

Section: Introductionmentioning

confidence: 99%

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Allosteric SHP2 Inhibition Increases Apoptotic Dependency on BCL2 and Synergizes with Venetoclax inFLT3-andKIT-Mutant AML

Popescu

Stahlhut

Tarver

et al. 2022

Preprint

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Mutations in receptor tyrosine kinases (RTKs) FLT3 and KIT are frequent and associated with poor outcomes in acute myeloid leukemia (AML). Although FLT3 inhibitors (FLT3i) are clinically effective, remissions are short-lived due to secondary resistance characterized by acquired mutations constitutively activating the RAS/MAPK pathway. Hereby, we report pre-clinical efficacy of co-targeting SHP2, a critical node in MAPK signaling, and BCL2 in RTK-driven AML. The allosteric SHP2 inhibitor RMC-4550 suppressed proliferation of AML cell lines with FLT3 and KIT mutations, including cell lines with acquired resistance to FLT3i. We demonstrate that SHP2 inhibition unveils an Achilles heel of AML, increasing apoptotic dependency on BCL2 via MAPK-dependent mechanisms, including upregulation of BMF and downregulation of MCL1. Consequently, RMC-4550 and venetoclax are synergistically lethal in FLT3- or KIT-mutant AML cell lines, and in clinically relevant xenograft models. Our results provide new mechanistic rationale and preclinical evidence for co-targeting SHP2 and BCL2 in RTK-driven AML.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Allosteric SHP2 Inhibition Increases Apoptotic Dependency on BCL2 and Synergizes with Venetoclax inFLT3-andKIT-Mutant AML

Popescu

Stahlhut

Tarver

et al. 2022

Preprint

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“…In contrast, a missense mutation N841K was detected in all quizartinib resistant cells (Figure 5b). FLT3 N841K has previously been detected in AML patients 21 , resides in the activation loop of FLT3 22 , and furthermore, mutation of the residue corresponding to N841 in the closely-related receptor tyrosine kinase KIT is activating 23 . This strongly suggests that N841K is a chief secondary mutation to ITD and is plausibly contributing to quizartinib resistance in this model by preventing efficiency of drug binding.…”

Section: Acquisition Of a Secondary Flt3 Mutation As A Key Driver Of ...mentioning

confidence: 99%

Unifying comprehensive genomics and transcriptomics in individual cells to illuminate oncogenic and drug resistance mechanisms

Zawistowski¹,

Salas-González²,

Morozova³

et al. 2022

Preprint

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Discovering genomic variation in the absence of information about transcriptional consequence of that variation or, conversely, a transcriptional signature without understanding underlying genomic contributions, hinders understanding of molecular mechanisms of disease. To assess this genomic and transcriptomic coordination, we developed a new chemistry and method, ResolveOME, to extract this information out of the individual cell. The workflow unifies template-switching full-transcript RNA-Seq chemistry and whole genome amplification (WGA), followed by affinity purification of first-strand cDNA and subsequent separation of the RNA/DNA fractions for sequencing library preparation. In the ResolveOME methodology we leverage the attributes of primary template-directed amplification (PTA)1 to enable accurate assessment of single-nucleotide variation as a DNA feature—not achieved with existing workflows to assess DNA + RNA information in the same cell.We demonstrated the validity of the technique in the context of two major phenomena in oncology: tumor heterogeneity (leading to cancer progression) and treatment resistance. Material from a primary patient breast cancer and an acute myeloid leukemia (AML) cell line, MOLM-13, was used to highlight multiomic biomarker paradigms enabled by this chemistry. Performance of the PTA-enabled genome amplification was largely unaffected by addition of RNA enrichment, with control WGS results showing > 95% genome coverage, precision > 0.99 and allele drop out < 15%. In the RNA fraction of the chemistry, we were able to routinely retrieve full-length transcripts that demonstrate a ratio of 1 for 5’/3’ bias, with increased coverage of intronic regions and 5’ regions that are indicative of novel transcripts, showing strength of the template switching mechanism to capture isoform information with sparsity rates < 75%. We find remarkable cellular variability of revealed biomarkers at both in the genome and transcriptome despite employing a relatively small number of individual cells. In our primary patient sample of ductal carcinoma in situ (DCIS)/invasive ductal carcinoma (IDC) we observed oncogenic PIK3CA driver mutations and prototypical DCIS copy number alterations binned into heterogenous single-cell classes of genomic lesions. Within our quizartinib-treated MOLM-13 cells, we identified multiple potential mechanisms of resistance within seemingly sporadic changes and were able to associate specific mutation, copy number and expression significantly correlated to treatment. In this latter scenario, the DNA arm of our combined workflow uncovered a secondary FLT3 (non-internal tandem duplication (ITD)) mutation as a candidate primary driver of resistance to drug while the RNA arm showed matched transcript upregulation of AXL signal transduction as well as enhancer factor modulation. Importantly, proximal candidate regulatory SNVs, outside of the CDS, were identified and associated to upregulated transcripts in cis. The study highlights that both the genome and transcriptome are dynamic, leading to a set of combinatorial alterations that affect cellular evolution and that fate can be identified through ResolveOME application to individual cells.

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“…Additionally, therapy with FLT3 inhibitors in patients with relapsed or refractory (R/R) AML who harbor FLT3 -ITD mutations has been shown to promote drug-resistant clonal populations that contain secondary, on-target mutations in FLT3 that confer resistance to multiple TKIs [ 9 , 10 ]. Because R/R AML is demonstrably polyclonal in nature, FLT3 TKIs may elicit clonal pressure to select for drug-resistant tumor cell populations with additional mutations that promote leukemic growth independent of the activation state of the FLT3 kinase [ 11 – 13 ], as well as for clones that lack FLT3 mutations entirely [ 11 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

Clinical outcomes in patients with relapsed/refractory FLT3-mutated acute myeloid leukemia treated with gilteritinib who received prior midostaurin or sorafenib

et al. 2022

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The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib is indicated for relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML), based on its observed superior response and survival outcomes compared with salvage chemotherapy (SC). Frontline use of FLT3 tyrosine kinase inhibitors (TKIs) midostaurin and sorafenib may contribute to cross-resistance to single-agent gilteritinib in the R/R AML setting but has not been well characterized. To clarify the potential clinical impact of prior TKI use, we retrospectively compared clinical outcomes in patients with R/R FLT3-mutated AML in the CHRYSALIS and ADMIRAL trials who received prior midostaurin or sorafenib against those without prior FLT3 TKI exposure. Similarly high rates of composite complete remission (CRc) were observed in patients who received a FLT3 TKI before gilteritinib (CHRYSALIS, 42%; ADMIRAL, 52%) and those without prior FLT3 TKI therapy (CHRYSALIS, 43%; ADMIRAL, 55%). Among patients who received a prior FLT3 TKI in ADMIRAL, a higher CRc rate (52%) and trend toward longer median overall survival was observed in the gilteritinib arm versus the SC arm (CRc = 20%; overall survival, 5.1 months; HR = 0.602; 95% CI: 0.299, 1.210). Remission duration was shorter with prior FLT3 TKI exposure. These findings support gilteritinib for FLT3-mutated R/R AML after prior sorafenib or midostaurin.

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Single-cell DNA sequencing reveals complex mechanisms of resistance to quizartinib

Abstract: Key Points Single-cell sequencing exposes previously unmeasurable complexity of tumor heterogeneity and clonal evolution on quizartinib. Single-cell sequencing reveals on- and off-target mechanisms of resistance to quizartinib, which can preexist therapy.

Cited by 23 publications

References 21 publications

Allosteric SHP2 Inhibition Increases Apoptotic Dependency on BCL2 and Synergizes with Venetoclax inFLT3-andKIT-Mutant AML

Allosteric SHP2 Inhibition Increases Apoptotic Dependency on BCL2 and Synergizes with Venetoclax inFLT3-andKIT-Mutant AML

Unifying comprehensive genomics and transcriptomics in individual cells to illuminate oncogenic and drug resistance mechanisms

Clinical outcomes in patients with relapsed/refractory FLT3-mutated acute myeloid leukemia treated with gilteritinib who received prior midostaurin or sorafenib

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