Where did we start?
Acute myeloid leukemia (AML) is an aggressive disease with recurrent mutations which lead to pathological proliferation of myeloid clones. One of the most frequent driver mutations occur in the Fms-like tyrosine kinase 3 as an internal tandem duplication (FLT3-ITD). For patients carrying this mutation, tyrosine kinase inhibitors (TKIs) such as midostaurin and gilteritinib are approved therapeutic options (1) (2). Nevertheless, therapy resistance remains a major problem, with high relapse rates and poor patient outcome. One concept of chemotherapy-resistance in leukemia is cell adhesion mediated drug resistance (CAM-DR) (3). As part of a medical doctoral project in the group “Epigenomics, Epitranscriptomics and Novel Therapy Approaches in AML” headed by Prof. Carsten Müller-Tidow at the University Hospital of Heidelberg, we investigated mechanisms underlying TKI drug resistance in FLT3-ITD mutated AML with a focus on CAM-DR.
What did we do/ What did we observe:
We generated two FLT3-ITD mutated midostaurin-resistant cell lines which we characterized by multilayered proteome analyses with the “Division Proteomics of Stem Cells and Cancer” headed by Prof. Jeroen Krijgsveld at the German Cancer Research Center (DKFZ) Heidelberg.
We were particularly interested in differentially expressed proteins with implications in cell adhesion and migration during induced resistance. We identified leupaxin (LPXN), a described regulator of cell adhesion and migration in different cancer entities (4), to be induced during emerging midostaurin resistance. We further found that the LPXN-interacting protein tyrosine kinase 2b (PTK2B) is the kinase that phosphorylates LPXN. PTK2B is a member of the focal adhesion kinase superfamily (5) and was concomitantly upregulated upon induced resistance.
Therefore, we assessed for the impact of inhibition of this novel axis in the context of AML and drug resistance. For this we used two different dual inhibitors of PTK2B and a family member, focal adhesion kinase (FAK), i.e. PF-431396 and defactinib. The inhibitor PF-431396 alone was able to revert resistant associated phenotypes such as enhanced cell migration, indicating altered niche interactions.
For a broader characterization of PTK2B/FAK inhibition effects we performed nascent proteome experiments of parental cells, as well as TKI resistant cells with and without PTK2B/FAK inhibitor treatment. In the majority of cases, treatment resulted in complete compensation of the effects on protein translation observed in resistant compared to parental cells.
Both PF-431396 and defactinib efficiency was specific for FLT3-mutated patient samples and cell lines. We combined both PTK2B/FAK inhibitors with other TKIs or daunorubicin and observed a strong synergy, again only in FLT3-mutated cell lines and patient samples. This synergy was particularly strong in our TKI resistant cell lines.
Finally, the combination of gilteritinib and defactinib was evaluated in vivo in collaboration with the group of Prof. Andreas Trumpp “Stem Cells and Cancer” at DKFZ. Mice treated with the gilteritinib/defactinib combination had lower leukemia burden and reached the time to leukemia symptom related endpoint later compared to mice treated with either drug alone.
What are our conclusions:
Our findings indicate that the addition of PF-431396 or defactinib to standard therapeutic regimens in FLT3-ITD AML targets important FLT3 mutation-associated niche interactions. Therefore, PTK2B/FAK inhibitors may be a beneficial add-on therapy for treatment of FLT3-ITD mutated patients, especially for those with prior relapse/refractoriness to TKIs. Further, an addition to first-line regimens might prevent the outgrowth of TKI-resistant clones at an early timepoint.
What should be studied in the future?
Future studies should focus on deciphering the precise mechanism and interplay between PTK2B/FAK inhibition and altered FLT3 signaling: e.g. why is the addition of a PTK2B/FAK inhibitor only beneficial in FLT3-mutated AML? Which precise pathway alterations result in PTK2B/FAK inhibitor susceptibility? Does this reveal additional therapeutic leverage points in FLT3-mutated AML?
In May 2021 the PTK2B/FAK inhibitor defactinib received breakthrough therapy designation status by the FDA for a combination treatment of ovarian cancer. Given the excellent tolerability of defactinib in the respective phase-I/II trials and the results of our study, the efficacy of the addition of defactinib to gilteritinib should be investigated in r/r FLT3-ITD AML patients in a clinical trial.
- Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer S, Bloomfield CD, et al. Midostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Engl J Med. 2017;377(5):454-64.
- Perl AE, Martinelli G, Cortes JE, Neubauer A, Berman E, Paolini S, et al. Gilteritinib or Chemotherapy for Relapsed or Refractory FLT3-Mutated AML. N Engl J Med. 2019;381(18):1728-40.
- Kim HN, Ruan Y, Ogana H, Kim YM. Cadherins, Selectins, and Integrins in CAM-DR in Leukemia. Front Oncol. 2020;10:592733.
- Dierks S, von Hardenberg S, Schmidt T, Bremmer F, Burfeind P, Kaulfuss S. Leupaxin stimulates adhesion and migration of prostate cancer cells through modulation of the phosphorylation status of the actin-binding protein caldesmon. Oncotarget. 2015;6(15):13591-606.
- Lipsky BP, Beals CR, Staunton DE. Leupaxin is a novel LIM domain protein that forms a complex with PYK2. J Biol Chem. 1998;273(19):11709-13.