To add even more nuance, even protected mode 16-bit Windows ran Windows apps in v86 mode. Essentially protected mode was a hypervisor you could launch from DOS and your actual Windows session would run on top of that. Before the VT-x extensions to x86-64, there was no more access to virtual-86 if you were running the processor in 64-bit long mode.

Considering that, it's no surprise that 16-bit Windows apps would no longer run on 64-bit Windows, especially since NTVDM was necessary to run them on 32-bit NT based versions of Windows anyway.