Before. For a startup, the lease is one of the main ways to show the funds are committed and the business is about to operate. Negotiate an early-termination or contingency clause if the landlord will agree, but do not expect to apply with nothing signed.
Commit first, then apply, then open
The sequence for a startup runs backwards from what caution suggests. The consular officer needs to see a business that is real and about to operate, and a lease, purchased equipment and a fit-out contract are what make it real. The couple therefore commits before knowing the outcome, and that exposure is what the at-risk test is measuring.
Reduce the exposure where the market allows: a landlord may accept a clause allowing termination if the visa is refused, an equipment supplier may accept a deposit with a documented refund schedule, and a contractor may agree to staged payments. Each of these must still leave real money at risk. Time the sale of the Canadian store so that the proceeds are available before the U.S.
commitments are due, then apply once the trail is complete. Canadians apply at a U.S. consulate for the E-2 visa; the interview date, not the paperwork, often sets the earliest opening day.