RAS G12C Inhibitors: Three Birds with One Stone.

SUMMARY: In this issue, Rubinson, Tanaka, and colleagues demonstrate that differences among G12C inhibitors rely on their ability to covalently bind not only G12C mutant KRAS but also NRAS and HRAS, proposing sotorasib as a potent NRAS G12C inhibitor. See related article by Rubinson et al., p. 727 (6).

Inhibition of NOTCH4 sensitizes FLT3/ITD acute myeloid leukemia cells to FLT3 tyrosine kinase inhibition.

Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition. Further investigation found increased NOTCH4 expression in FLT3/ITD AML cell lines and primary patient samples. Inhibition of NOTCH4 by shRNA knockdown, CRISPR-Cas9-based knockout or γ-secretase inhibitors synergized with FLT3 TKIs to kill FLT3/ITD AML cells in vitro. NOTCH4 inhibition sensitized TKI-resistant FLT3/ITD cells to FLT3 TKI inhibition. The combination reduced phospho-ERK and phospho-AKT, indicating inhibition of MAPK and PI3K/AKT signaling pathways. It also led to changes in expression of genes involved in regulating cell cycling, DNA repair and transcription. A patient-derived xenograft model showed that the combination reduced both the level of leukemic involvement of primary human FLT3/ITD AML cells and their ability to engraft secondary recipients. In summary, these results demonstrate that NOTCH4 inhibition synergizes with FLT3 TKIs to eliminate FLT3/ITD AML cells, providing a new therapeutic target for AML with FLT3/ITD mutations.

FLT3 tyrosine kinase inhibitors synergize with BCL-2 inhibition to eliminate FLT3/ITD acute leukemia cells through BIM activation.

Tyrosine kinase inhibitors (TKIs) targeting FLT3 have shown activity but when used alone have achieved limited success in clinical trials, suggesting the need for combination with other drugs. We investigated the combination of FLT3 TKIs (Gilteritinib or Sorafenib), with Venetoclax, a BCL-2 selective inhibitor (BCL-2i), on FLT3/ITD leukemia cells. The combination of a FLT3 TKI and a BCL-2i synergistically reduced cell proliferation and enhanced apoptosis/cell death in FLT3/ITD cell lines and primary AML samples. Venetoclax also re-sensitized FLT3 TKI-resistant cells to Gilteritinib or Sorafenib treatment, mediated through MAPK pathway inhibition. Gilteritinib treatment alone dissociated BIM from MCL-1 but increased the binding of BIM to BCL-2. Venetoclax treatment enhanced the binding of BIM to MCL-1 but dissociated BIM from BCL-2. Treatment with the drugs together resulted in dissociation of BIM from both BCL-2 and MCL-1, with an increased binding of BIM to the cell death mediator BAX, leading to increased apoptosis. These findings suggest that Venetoclax mitigates the unintended pro-survival effects of FLT3 TKI mainly through the dissociation of BIM and BCL-2 and also decreased BIM expression. This study provides evidence that the addition of BCL-2i enhances the effect of FLT3 TKI therapy in FLT3/ITD AML treatment.

Acetylcholinesterase is not a generic marker of extracellular vesicles.

Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum "extra-depletion" protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.

Combination of ATO with FLT3 TKIs eliminates FLT3/ITD+ leukemia cells through reduced expression of FLT3.

Acute myeloid leukemia (AML) patients with FLT3/ITD mutations have a poor prognosis. Monotherapy with selective FLT3 tyrosine kinase inhibitors (TKIs) have shown transient and limited efficacy due to the development of resistance. Arsenic trioxide (ATO, As2O3) has been proven effective in treating acute promyelocytic leukemia (APL) and has shown activity in some cases of refractory and relapsed AML and other hematologic malignances. We explored the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3/ITD+ leukemias. The combination of FLT3 TKIs with ATO showed synergistic effects in reducing proliferation, viability and colony forming ability, and increased apoptosis in FLT3/ITD+ cells and primary patient samples. In contrast, no cooperativity was observed against wild-type FLT3 leukemia cells. ATO reduced expression of FLT3 RNA and its upstream transcriptional regulators (HOXA9, MEIS1), and induced poly-ubiquitination and degradation of the FLT3 protein, partly through reducing its binding with USP10. ATO also synergizes with FLT3 TKIs to inactivate FLT3 autophosphorylation and phosphorylation of its downstream signaling targets, including STAT5, AKT and ERK. Furthermore, ATO combined with sorafenib, a FLT3 TKI, in vivo reduced growth of FLT3/ITD+ leukemia cells in NSG recipients. In conclusion, these results suggest that ATO is a potential candidate to study in clinical trials in combination with FLT3 TKIs to improve the treatment of FLT3/ITD+ leukemia.

Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit.

Obscurins are a family of giant cytoskeletal proteins, originally identified in striated muscles where they have structural and regulatory roles. We recently showed that obscurins are abundantly expressed in normal breast epithelial cells where they play tumor and metastasis suppressing roles, but are nearly lost from advanced stage breast cancer biopsies. Consistent with this, loss of giant obscurins from breast epithelial cells results in enhanced survival and growth, epithelial to mesenchymal transition (EMT), and increased cell migration and invasion in vitro and in vivo. In the current study, we demonstrate that loss of giant obscurins from breast epithelial cells is associated with significantly increased phosphorylation and subsequent activation of the PI3K signaling cascade, including activation of AKT, a key regulator of tumorigenesis and metastasis. Pharmacological and molecular inhibition of the PI3K pathway in obscurin-depleted breast epithelial cells results in reversal of EMT, (re)formation of cell-cell junctions, diminished mammosphere formation, and decreased cell migration and invasion. Co-immunoprecipitation, pull-down, and surface plasmon resonance assays revealed that obscurins are in a complex with the PI3K/p85 regulatory subunit, and that their association is direct and mediated by the obscurin-PH domain and the PI3K/p85-SH3 domain with a KD of ~50 nM. We therefore postulate that giant obscurins act upstream of the PI3K cascade in normal breast epithelial cells, regulating its activation through binding to the PI3K/p85 regulatory subunit.